Production and use of red blood cells

ABSTRACT

The present disclosure relates to the production of red blood cells from hematopoietic stem cells, by differentiating such cells in the presence of a protein that induces cell survival and proliferation.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/643,133, filed Jul. 6, 2017, which is a division of U.S. patentapplication Ser. No. 13/797,648, filed Mar. 12, 2013, which claims thebenefit of U.S. Provisional Application No. 61/776,732, filed Mar. 11,2013, each of which is hereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 691772000900SeqList.txt,date recorded: Mar. 11, 2013, size: 9 KB)

FIELD

The present disclosure relates to new methods of producing mature redblood cells from hematopoietic stem cells in vitro, and theirtherapeutic and diagnostic use in vivo.

BACKGROUND

Transfusion of red blood cells (RBCs) is routinely used for manyclinical and surgical applications. On average, 39,000 units of bloodare needed every day, and data from 2004 indicate that 29 million unitsof blood were transfused in one year (American Association of BloodBanks website). This procedure has single-handedly saved many lives overthe past 60 years. The demand for such transfusions continues toincrease with advances in medical treatments and an aging population.

In addition to the traditional clinical settings that have benefitedfrom the availability of red blood cell transfusion, such as surgery andtreatment of trauma patients, there are a number of unique instances inwhich red blood cell transfusion would change the standard of care. Forexample, there are a number of rare phenotypes of RBCs in patients ofAfro-Caribbean descent (Douay et al., Transfusion Medicine Reviews 21,91-100, 2007). They are considered rare phenotypes, due to the lack ofantigens such as H or ABO blood groups. Such patients can develop aneutralizing antibody response to ABO blood group antigens, renderingthem ineligible for RBC transfusions. In fact, such patients mustreceive transfusions from an identical source to avoid a neutralizingantibody response, posing tremendous challenges in cases where repeatedred blood cell transfusions are required (e.g., sickle cell patients,etc.).

Additionally, patients who suffer from a variety of antibody-basedautoimmune diseases and experience autoimmune hemolytic anemia may alsobenefit from red blood cell transfusions. However, this presents achallenge in finding a donor, or limited set of donors whose RBCs arecompatible with the patients' autoantibodies. In essence, these patientsexperience the same challenges as those with rare blood phenotypes.

Moreover, patients who suffer from hemoglobinopathies and thalassemiashave congenic mutations that result in a shorter life span for theirRBCs. While the idea of improving the life and health of these patientswith blood transfusions is an old one, the frequency of transfusionsrequired presents a major problem. The average lifespan of RBCs from ahealthy donor is 28 days. The number of transfusions required for thesepatients is large, frequent, and poses a significantly increased risk ofiatrogenic infection. The ability to generate RBCs in vitro and toprovide transfusions of synchronized RBCs with a mean lifespan of 120days would greatly reduce the number of transfusions required for thesepatients and truly improve their quality of life.

The issue of lifespan of RBCs collected from donors is also important inthe context of traditional clinical use of RBCs for trauma and surgicalprocedures. The storage of RBC concentrates for up to one month mayresult in an RBC population that requires at least 24 hours to recoverits ability to transport oxygen. In addition, a number of necrotic RBCsin those concentrates could trigger an inflammatory response in therecipient, along with the complications that arise from such aninflammatory response. The ability to generate a constant supply of RBCsin vitro would allow health professionals to anticipate and to meet thedemands for fresh RBCs, and would also eliminate the need for long-termstorage of RBC concentrates

Another problem with red blood cell transfusions is the increasingdifficulty in providing red blood cell transfusions. The reasons forthis increasing difficulty include a steady drop in the supply ofdonated blood that is eligible for transfusion due to the increasednumber of infectious agents that have been shown to be transmittedthrough blood transfusions, the failure of hemoglobin and oxygentransporters (perflourocarbons) to show efficacy as RBC alternatives inthe clinical setting, and recent complications associated witherythropoietin (EPO) usage. Ready access to a continuous supply of RBCprogenitors that could generate a defined RBC product for transfusionwould alter the practice in the clinic and render blood transfusion asafer and more extensively used procedure. However, such an approachmust be able to provide a supply of RBCs that is safe, effective, anduniversal.

While some initial attempts have been made to derive RBCs in vitro fromprimary hematopoietic stem cells (derived from bone marrow, cord blood,or peripheral blood) or embryonic stem cells, to date they have beenunsuccessful for a variety of reasons including one or more of expense,duration of protocol, multiple steps, use of feeder cells or serum,labor intensiveness, low yield, or failure to fully differentiate tomature, anucleated red blood cells. Attempts to generate RBCs in vitroinclude methods starting from primary hemotopoietic stem cells(Neildez-Nguyen et al., Nat Biotech 20, 467-72, 2002), and embryonicstem cells (Lu et al., Blood. 2008 Dec. 1; 112(12):4475-84; Lu et al.,Regen Med 3, 693-704, 2008. These approaches also don't generally allowfor a defined and continuous source of RBC progenitors.

Citation of the above documents and studies is not intended as anadmission that any of the foregoing is pertinent prior art. Allstatements as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the contents of these documents.

BRIEF SUMMARY

Accordingly, there is a need for improved approaches for in vitroproduction of fully mature human red blood cells. The present disclosureprovides novel methods of producing a red blood cell (RBC) population byculturing hematopoietic stem cells (HSCs) in the presence of one or morerecombinant protein, such as an exogenous protein, that induces one ormore of cell survival or proliferation, EPO, and optionally, IL-3.Advantageously, these methods produce mature anucleated red blood cellsin about 10 days that exhibit an adult red blood cell phenotype thatincludes, without limitation, expression of Glycophrin A (GPA),increased levels of adult hemoglobin, decreased levels of CD71(transferrin receptor), and decreased levels of fetal hemoglobin.Moreover, production of the RBCs from conditionally immortalized humanlong-term HSCs that can be passaged indefinitely in vitro,cryopreserved, and recovered, allows for the continuous production offully differentiated red blood cells from a defined, well-characterized,source. Additionally, RBCs produced by the novel methods of the presentdisclosure may also contain and/or express one or more recombinantprotein of interest that can be used to treat a subject in need thereof.

Accordingly, certain aspects of the present disclosure relate to amethod for producing a population of mature red blood cells fromhematopoietic stem cells, by: culturing the hematopoietic stem cells inthe presence of EPO and one or more first recombinant protein, orbiologically active fragment thereof, that promotes cell survival and/orproliferation; under conditions that induce differentiation of thehematopoietic stem cells to mature red bloods cells, thereby producing apopulation of mature red blood cells. In some embodiments, thehematopoietic stem cells are conditionally immortalized hematopoieticstem cells. In some embodiments that may be combined with any of thepreceding embodiments, the hematopoietic stem cells are proteintransduced hematopoietic stem cells, and the one or more firstrecombinant protein, or biologically active fragment thereof, is anexogenous protein. In some embodiments that may be combined with any ofthe preceding embodiments, the hematopoietic stem cells are transgenichematopoietic stem cells. In some embodiments that may be combined withany of the preceding embodiments, the one or more first recombinantprotein, or biologically active fragment thereof, is one or morepolypeptide selected from a MYC polypeptide, an ICN-1 polypeptide,homologues thereof, and biologically active fragments thereof. In someembodiments that may be combined with any of the preceding embodiments,the MYC polypeptide is one or more MYC polypeptide selected from n-Myc,c-Myc, 1-Myc, v-Myc, and s-Myc. In some embodiments that may be combinedwith any of the preceding embodiments, one or more of the one or morefirst recombinant protein, or biologically active fragment thereof,contains a protein transduction domain. In some embodiments that may becombined with any of the preceding embodiments, the protein transductiondomain is one or more protein transduction domain selected from TAT,VPR, and EPTD. In some embodiments that may be combined with any of thepreceding embodiments, the one or more first recombinant protein, orbiologically active fragment thereof, is TAT-MYC. In some embodimentsthat may be combined with any of the preceding embodiments, the one ormore first recombinant protein, or biologically active fragment thereof,is provided as a bolus. In some embodiments that may be combined withany of the preceding embodiments, the one or more first recombinantprotein is provided as a bolus about every 24 hours, about every 48hours, or about every 72 hours. In some embodiments that may be combinedwith any of the preceding embodiments, the method further includesculturing the hematopoietic stem cells in the presence of IL-3. In someembodiments that may be combined with any of the preceding embodiments,the method further includes culturing the hematopoietic stem cells inthe absence of feeder cells and serum. In some embodiments that may becombined with any of the preceding embodiments, the method furtherincludes culturing the population of mature red blood cells in thepresence of one or more second recombinant protein, or biologicallyactive fragment thereof, that inhibits apoptosis. In some embodimentsthat may be combined with any of the preceding embodiments, the one ormore second recombinant protein, or biologically active fragmentthereof, contains one or more Bcl-2 homology domains. In someembodiments that may be combined with any of the preceding embodiments,the one or more Bcl-2 homology domains are one or more Bcl-2 homologydomains selected from BH1, BH2, BH3, and BH4. In some embodiments thatmay be combined with any of the preceding embodiments, the one or moresecond recombinant protein is one or more of Bcl-2, Bcl-w, Bcl-X,Bcl-XL, or Mcl-1. In some embodiments that may be combined with any ofthe preceding embodiments, the one or more second recombinant protein,or biologically active fragment thereof, is Bcl-2. In some embodimentsthat may be combined with any of the preceding embodiments, one or moreof the one or more second recombinant protein, or biologically activefragment thereof, contains a protein transduction domain. In someembodiments that may be combined with any of the preceding embodiments,the protein transduction domain is one or more protein transductiondomains selected from TAT, VPR, and EPTD. In some embodiments that maybe combined with any of the preceding embodiments, the one or moresecond recombinant protein, or biologically active fragment thereof, isTAT-Bcl-2. In some embodiments that may be combined with any of thepreceding embodiments, the hematopoietic stem cells further contain oneor more recombinant protein of interest, or biologically active fragmentthereof. In some embodiments that may be combined with any of thepreceding embodiments, the one or more recombinant protein of interest,or biologically active fragment thereof, is an exogenous protein, orbiologically active fragment thereof. In some embodiments that may becombined with any of the preceding embodiments, the hematopoietic stemcells contain one or more transgenes that encode one or more proteinsselected from the one or more first recombinant protein, or biologicallyactive fragment thereof; the one or more second recombinant protein, orbiologically active fragment thereof; and one or more recombinantprotein of interest, or biologically active fragment thereof. In someembodiments that may be combined with any of the preceding embodiments,the expression or function of one or more of the one or more firstrecombinant protein, or biologically active fragment thereof; the one ormore second recombinant protein, or biologically active fragmentthereof; or one or more recombinant protein of interest, or biologicallyactive fragment thereof is controllable. In some embodiments that may becombined with any of the preceding embodiments, the expression orfunction of one or more of the one or more first recombinant protein, orbiologically active fragment thereof; the one or more second recombinantprotein, or biologically active fragment thereof; or one or morerecombinant protein of interest, or biologically active fragment thereofis inducible. In some embodiments that may be combined with any of thepreceding embodiments, one or more of the one or more transgenes encodean antibiotic responsive element or a hormone responsive element. Insome embodiments that may be combined with any of the precedingembodiments, the antibiotic responsive element or the hormone responsiveelement is one or more responsive element selected from an estrogenresponse element, a gonadotropin response element, or a tetracyclineresponse element, and a glucocorticoid response element. In someembodiments that may be combined with any of the preceding embodiments,the one or more transgenes over-express one or more one or more proteinsselected from the one or more first recombinant protein, or biologicallyactive fragment thereof; the one or more second recombinant protein, orbiologically active fragment thereof; and one or more recombinantprotein of interest, or biologically active fragment thereof. In someembodiments that may be combined with any of the preceding embodiments,the production of the population of mature red blood cells isaccelerated by at least 45% compared to production of a population ofred blood cells from a primary stem cell cultured in the presence ofIL-3 and EPO for eight days, then in the presence of feeder cells andEPO for three days, and finally in the presence of feeder cells alonefor 10 days. In some embodiments that may be combined with any of thepreceding embodiments, the population of mature red blood cells isproduced in about 7 to 14 days. In some embodiments that may be combinedwith any of the preceding embodiments, the population of mature redblood cells exhibits one or more characteristics selected from apopulation of mature red blood cells, where about 40% to about 100% ofthe cells are anucleated; a population of mature red blood cells, whereabout 40% to about 100% of the cells express GPA; a population of maturered blood cells, where about 40% to about 100% of the cells expressadult hemoglobin; a population of mature red blood cells, where about40% to about 100% of the cells exhibit decreased levels of CD71expression; a population of mature red blood cells, where about 40% toabout 100% of the cells exhibit decreased levels of fetal hemoglobinexpression. In some embodiments that may be combined with any of thepreceding embodiments, the hematopoietic stem cells are humanhematopoietic stem cells. In some embodiments that may be combined withany of the preceding embodiments, the hematopoietic stem cells wereisolated from a patient with a rare blood type. In some embodiments thatmay be combined with any of the preceding embodiments, the patient hasan autoimmune condition. In some embodiments that may be combined withany of the preceding embodiments, the hematopoietic stem cells wereproduced from embryonic stem cells or induced pluripotent stem cells. Insome embodiments that may be combined with any of the precedingembodiments, the population of mature red blood cells is a population ofhuman cells. In some embodiments that may be combined with any of thepreceding embodiments, the population of mature red blood cells has oneor more blood types selected from A⁺, A⁻, B⁺, B⁻, AB⁺, AB⁻, O⁺, and O⁻.In some embodiments that may be combined with any of the precedingembodiments, the population of mature red blood cells is a rare bloodtype. In some embodiments that may be combined with any of the precedingembodiments, the population of mature red blood cells is a population ofnon-human animal cells.

Other aspects of the present disclosure relate to a population of invitro differentiated mature red blood cells, containing anucleated redblood cells expressing GPA, expressing adult hemoglobin, exhibitingdecreased levels of CD71 expression, and exhibiting decreased levels offetal hemoglobin expression, where: about 40% to about 100% of the redblood cells in the population are anucleated; about 40% to about 100% ofthe red blood cells in the population express GPA; about 40% to about100% of the red blood cells in the population express adult hemoglobin;about 40% to about 100% of the red blood cells in the population exhibitdecreased levels of CD71 expression; and about 40% to about 100% of thered blood cells in the population exhibit decreased levels of fetalhemoglobin expression. In some embodiments that may be combined with anyof the preceding embodiments, the red blood cells contain one or morerecombinant protein of interest. In some embodiments that may becombined with any of the preceding embodiments, the red blood cells havea rare blood type. In some embodiments that may be combined with any ofthe preceding embodiments, the red blood cells are human red bloodcells. In some embodiments that may be combined with any of thepreceding embodiments, the red blood cells are non-human animal redblood cells. In some embodiments that may be combined with any of thepreceding embodiments, the population of in vitro differentiated maturered blood cells is produced by any methods of producing a population ofmature red blood cells from hematopoietic stem cells of any of thepreceding embodiments.

Other aspects of the present disclosure relate to a pharmaceuticalcomposition containing: a population of in vitro differentiated maturered blood cells, where about 40% to about 100% of the red blood cells inthe population are anucleated, about 40% to about 100% of the red bloodcells in the population express GPA, about 40% to about 100% of the redblood cells in the population express adult hemoglobin, about 40% toabout 100% of the red blood cells in the population exhibit decreasedlevels of CD71 expression, and about 40% to about 100% of the red bloodcells in the population exhibit decreased levels of fetal hemoglobinexpression; and one or more pharmaceutically acceptable excipients. Insome embodiments that may be combined with any of the precedingembodiments, where the composition further contains one or morerecombinant protein of interest, or biologically active fragmentthereof. In some embodiments that may be combined with any of thepreceding embodiments, the population of red blood cells contains one ormore recombinant protein of interest, or biologically active fragmentthereof. In some embodiments that may be combined with any of thepreceding embodiments, the one or more recombinant protein of interest,or biologically active fragment thereof, is an exogenous protein. Insome embodiments that may be combined with any of the precedingembodiments, the red blood cells have a rare blood type. In someembodiments that may be combined with any of the preceding embodiments,the red blood cells are human red blood cells. In some embodiments thatmay be combined with any of the preceding embodiments, the red bloodcells are non-human animal red blood cells. In some embodiments that maybe combined with any of the preceding embodiments, the population of invitro differentiated mature red blood cells is produced by any methodsof producing a population of mature red blood cells from hematopoieticstem cells of any of the preceding embodiments.

Other aspects of the present disclosure relate to a method of treatment,prevention, or diagnosis of a disease or disorder characterized by adeficiency of anucleated red blood cells by: providing to a subject inneed thereof a population of in vitro differentiated mature red bloodcells, where about 40% to about 100% of the red blood cells in thepopulation are anucleated, about 40% to about 100% of the red bloodcells in the population express GPA, about 40% to about 100% of the redblood cells in the population express adult hemoglobin, about 40% toabout 100% of the red blood cells in the population exhibit decreasedlevels of CD71 expression, and about 40% to about 100% of the red bloodcells in the population exhibit decreased levels of fetal hemoglobinexpression. In some embodiments that may be combined with any of thepreceding embodiments, the subject is a human. In some embodiments thatmay be combined with any of the preceding embodiments, the subject is anon-human animal. In some embodiments that may be combined with any ofthe preceding embodiments, the population of red blood cells containsone or more recombinant protein of interest, or a biologically activefragment thereof. In some embodiments that may be combined with any ofthe preceding embodiments, the one or more recombinant protein ofinterest, or biologically active fragment thereof, is an exogenousprotein. In some embodiments that may be combined with any of thepreceding embodiments, the population of in vitro differentiated maturered blood cells are produced by any methods of producing a population ofmature red blood cells from hematopoietic stem cells of any of thepreceding embodiments.

Other aspects of the present disclosure relate to a method for extendingthe half-life of a population of mature red blood cells in vitro by:maintaining the population of red blood cells in a media containing oneor more exogenous polypeptide, homologues thereof or biologically activefragments thereof, that inhibit apoptosis. In some embodiments that maybe combined with any of the preceding embodiments, the one or moreexogenous polypeptide, homologues thereof or biologically activefragments thereof, contains one or more Bcl2 homology domains. In someembodiments that may be combined with any of the preceding embodiments,the one or more Bcl-2 homology domains are one or more Bcl-2 homologydomains selected from BH1, BH2, BH3, and BH4. In some embodiments thatmay be combined with any of the preceding embodiments, the one or moreexogenous polypeptide, homologues thereof or biologically activefragments thereof, is one or more of Bcl-2, Bcl-w, Bcl-X, Bcl-XL, orMcl-1. In some embodiments that may be combined with any of thepreceding embodiments, the one or more exogenous polypeptide, homologuesthereof or biologically active fragments thereof, is Bcl-2. In someembodiments that may be combined with any of the preceding embodiments,one or more of the one or more exogenous polypeptide, homologues thereofor biologically active fragments thereof, contains a proteintransduction domain. In some embodiments that may be combined with anyof the preceding embodiments, the protein transduction domain is one ormore protein transduction domains selected from TAT, VPR, and EPTD. Insome embodiments that may be combined with any of the precedingembodiments, the one or more exogenous polypeptide, homologues thereofor biologically active fragments thereof, is TAT-Bcl-2. In someembodiments that may be combined with any of the preceding embodiments,the population of mature red blood cells is produced by any methods ofproducing a population of mature red blood cells from hematopoietic stemcells of any of the preceding embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict a general approach for the generation of ctlt-HSCcell lines. FIG. 1A depicts a schematic representation of retroviralconstructs used to transduce primary murine HSC cells with MYC-ER andBcl-2. FIG. 1B depicts flow cytometric analyses of HSCs obtained from5-fluorouracil treated donors following transduction with pMIG-MYC-ERand pMIG-Bcl-2. FIG. 1C depicts kinetics of leukemogenesis in micetransplanted with the transduced HSCs shown in FIG. 1B. FIG. 1D depictsFACS analysis of a ctlt-HSC soon after recovery from the bone marrow ofleukemic mice. FIG. 1E depicts FACS analysis of a ctlt-HSC cell linethat has been established. FIG. 1F depicts FACS analysis of stem cellmarkers in normal, unmanipulated Lt-HSCs from the bone marrow of wildtype C57/BL6 mice.

FIGS. 2A-2D depict the characterization of mature immune cells thatarise following transplantation of ctlt-HSCs. FIG. 2A depicts thefrequency of cells derived from ctlt-HSCs in lymphoid tissues aftertransplantation. FIG. 2B depicts the detection of GFP⁺ myeloid lineagecells in the bone marrow. FIG. 2C depicts the analysis of peripheral,mature lymphocytes in the spleen. FIG. 2D depicts the analysis ofperipheral, mature lymphocytes in transplant recipient mice followingthe second serial passage of ctlt-HSCs.

FIGS. 3A-3I depict the surface phenotype of three human ctlt-HSC celllines. FIG. 3A, FIG. 3B, and FIG. 3C show the CD34⁺ fraction oftransduced (i.e., GFP⁺) HSCs from three established human ctlt-HSC celllines. FIG. 3D, FIG. 3E, and FIG. 3F show the c-kit⁺ fraction oftransduced (i.e., GFP⁺) HSCs from three established human ctlt-HSC celllines. FIG. 3G depicts that the transduced (i.e., GFP⁺) HSCs from theestablished human ctlt-HSC cell lines do not express CD45. FIG. 3Hdepicts that the transduced (i.e., GFP⁺) HSCs from the established humanctlt-HSC cell lines do not express Flk-2. FIG. 3I depicts that thetransduced (i.e., GFP⁺) HSCs from the established human ctlt-HSC celllines do not express CD150.

FIGS. 4A-4D depict the differentiation of human ctlt-HSC cells linesinto mature lymphoid cells in NOD/SCID/β2M^(−/−) mice. FIG. 4A depictsdata obtained from a control mouse (no transplant). FIG. 4B, FIG. 4C,and FIG. 4D show data obtained from a transplant recipient mouse thatpresented mature human lymphoid cells in the peripheral blood.

FIGS. 5A-5F depict human red blood cells generated in vitro using humanctlt-HSCs as the source. FIG. 5A depicts H and E staining of mouseperipheral blood. FIG. 5B depicts H and E staining of primary humanfetal cord blood. FIG. 5C, FIG. 5D, and FIG. 5E show H and E staining ofthree conditionally transformed fetal cord blood cell lines that weretreated for 12 days with IL-3 and EPO. FIG. 5F shows a magnified view ofthe cells from FIG. 5E to show red blood cell morphology.

FIG. 6 depicts that in vitro generated human RBCs can rescue mice fromchemically induced lethal anemia.

FIG. 7 depicts that in vitro generated human RBCs can rescue mice fromhemorrhagic shock.

FIGS. 8A and 8B depict the expression and purification of recombinantTAT-MYC and TAT-Bcl-2 protein for direct transduction of protein intocells. FIG. 8A depicts the TAT-MYC. FIG. 8B depicts TAT-Bcl-2.

FIGS. 9A-9D depict FACS analysis showing the development of murineprotein transduced long-term HSC cell lines (ptlt-HSCs). FIG. 9A depictsunstained cells. FIG. 9B depicts staining with antibodies to Sca1 andc-Kit. FIG. 9C depicts staining with antibodies to B220 and CD3. FIG. 9Ddepicts the surface phenotype of the resulting ptlt-HSC line.

FIGS. 10A-10D depict FACS analysis showing reconstitution of lymphoidcompartment in Rag-1^(−/−) mice by a murine ptlt-HSC cell line. FIG. 10Adepicts unstained cells. FIG. 10B depicts staining for B cell markersB220 and IgM. FIG. 10C depicts staining for T cell markers CD4 and TCRβ.FIG. 10D depicts staining for T cell markers CD8 and TCRβ.

FIGS. 11A-11C depict the development of mature murine red blood cellsfrom ptlt-HSCs in vitro. FIG. 11A depicts H and E staining ofundifferentiated murine ptlt-HSCs under a 40×objective. FIG. 11B depictsH and E staining of red blood cells derived from murine ptlt-HSCs undera 40× objective. FIG. 11C depicts H and E staining of red blood cellsderived from murine ptlt-HSCs under a 100× objective.

FIGS. 12A and 12B depict cord blood-derived HSC expansion in vitro. FIG.12A depicts staining with antibodies to CD38 and CD34 after culturingthe HSCs for 3 days in medium containing TAT-MYC and TAT-Bcl-2. FIG. 12Bdepicts staining with antibodies to CD38 and CD34 after culturing theHSCs for 14 days in medium containing TAT-MYC and TAT-Bcl-2.

FIGS. 13A-13C depict the induction of red blood cell differentiation invitro. FIG. 13A depicts unstained HSC cells from FIG. 12 after 4 days ofculturing in medium containing IL-3 and EPO. FIG. 13B depicts stainingwith antibodies to GPA and CD71 after culturing the HSC cells from FIG.12 for 4 days in neutral medium. FIG. 13C depicts staining withantibodies to GPA and CD71 after culturing the HSC cells from FIG. 12for 4 days in medium containing IL-3 and EPO.

FIGS. 14A-14C depict dynamic analysis of red blood cell differentiationmarkers in vitro. FIG. 14A depicts staining with antibodies to GPA,CD71, and fetal hemoglobin of mature human RBC cells (+ Control). FIG.14B depicts staining with antibodies to GPA, CD71, and fetal hemoglobinafter 5 days of culturing the HSC cells from FIG. 12 in mediumcontaining IL-3 and EPO. FIG. 14C depicts staining with antibodies toGPA, CD71, and fetal hemoglobin after 9 days of culturing the HSC cellsfrom FIG. 12 in medium containing IL-3 and EPO.

FIGS. 15A and 15B depict histological analysis of enucleation inculture. FIG. 15A depicts H and E staining of cells from FIG. 12 after 3days of culturing in medium containing IL-3 and EPO. FIG. 15B depicts Hand E staining of cells from FIG. 12 after 7 days of culturing in mediumcontaining IL-3 and EPO.

FIGS. 16A and 16B depict human red blood cell production in agas-permeable bag. FIG. 16A depicts red blood cells differentiated fromconditionally immortalized ptlt-HSCs after culturing in mediumcontaining IL-3 and EPO for 4 days. FIG. 16B depicts H and E staining ofred blood cells, showing maturation and enucleation of the red bloodcells.

FIGS. 17A-17E depict mouse red blood cell production from bone marrowcells. FIG. 17A depicts H and E staining of untreated (control) mousehematopoietic stem cells from bone marrow. FIG. 17B depicts H and Estaining of mouse hematopoietic stem cells from bone marrow treated withIL3 and EPO, but not Tat-Myc or Tat-Bcl-2. FIG. 17C depicts H and Estaining of mouse hematopoietic stem cells from bone marrow treated withIL3, EPO and Tat-Myc, but not Tat-Bcl-2. FIG. 17D depicts H and Estaining of mouse hematopoietic stem cells from bone marrow treated withIL3, EPO, and Tat-Bcl-2, but not Tat-Myc. FIG. 17E depicts H and Estaining of mouse hematopoietic stem cells from bone marrow treated withIL3, EPO, Tat-Bcl-2, and Tat-Myc.

FIGS. 18A-18E depict the generation and in vitro characterization of Tatfusion proteins. FIG. 18A depicts a graphical representation of Tat-Mycand Tat-Bcl-2 fusion proteins including the location of the in frameprotein transduction domain of HIV-1 Tat and the V5 and 6×His tags. FIG.18B depicts recombinant proteins following purification from E. coli,separation by SDS-PAGE, and staining with Coomassie. FIG. 18C depicts alawn of confluent 3T3 cells exposed to purified recombinant Tat-Myc,Tat-Bcl-2, or left untreated (NT) for two hours, and then fixed andstained with a monoclonal antibody to V5 and with a Hoechst 9934 nuclearstain. The Tat-Myc protein largely localized to the nuclear region inthis timeframe, whereas the Tat-Bcl-2 remained in the cytoplasmic andperinuclear space. FIG. 18D depicts an SDS-PAGE and western blotanalysis (monoclonal antibodies to V5 and (3-actin) of human cord bloodderived HSCs pulsed with a single exposure of Tat-Myc for 1 hours,washed, and then lysed (at the indicated time points) to separate theplasma membrane and cytoplasmic fraction from the nuclear fraction. FIG.18E depicts a SDS-PAGE and western blot analysis (monoclonal antibodiesto V5 and (3-actin) of the nuclear fraction of human cord blood derivedHSCs pulsed with a single exposure of Tat-Myc for 2 hours, washed, andthen lysed (at the indicated time points) to separate the plasmamembrane and cytoplasmic fraction from the nuclear fraction. The bulk ofthe protein is lost between 24 and 48 hours. There is no detectableprotein left at any point after 72 hours.

FIGS. 19A-19E depict a graphical representation of the expansion ofhuman cord blood cell-derived HSCs with Tat-Myc and Tat-Bcl-2. FIG. 19Adepicts a graphical representation of a FACS analysis of the surfacephenotype of the human cord blood cells expanded in vitro for 14 days(Top panels cytokine cocktail only; Bottom panels cytokine cocktailsupplemented with Tat-Myc and Tat-Bcl-2). FIG. 19B depicts a graphicalrepresentation of the kinetics of CD34+ cells expansion in vitro underboth sets of conditions. FIG. 19C depicts the images of three differentcolony types developed in methylcellulose assays under conditions thatsupport myeloerythroid differentiation, derived from human ptlt-HSCs.FIG. 19D depicts a graphical representation of the quantification ofeach colony type that was observed in methylcellulose cultures seededwith either 10³ cord blood cells cultured with a cytokine cocktail(black bars), 10³ cord blood cells cultured with a cytokine cocktailsupplemented with Tat-Myc and Tat-Bcl-2 (dark grey bars), or 10⁴ freshun-manipulated cord blood cells (light grey bars). FIG. 19E depicts agraphical representation of the quantification of the number of coloniesobserved in methylcellulose cultures upon replating of the cells shownin FIG. 19D.

FIGS. 20A-20G depict a graphical representation of the functionalanalysis of human cord blood derived protein-transduced long term(ptlt)-HSC in vivo. FIG. 20A depicts a graphical representation of aFACS analysis of the bone marrow of cohorts of sublethally irradiatedNSG mice given transplants of 10⁶ cord blood cells expanded in vitro ina cocktail of cytokines (first panel; FCB), or expanded in a cocktail ofcytokines supplemented with Tat-Myc and Tat-Bcl-2 (second panel; FCBTMTB), or 5×10⁶ fresh un-manipulated cord blood cells (third panel;Fresh FCB). FIG. 20B depicts a graphical representation of a FACSanalysis of bone marrow, spleen and thymus cells from the xenochimaericmice. All cells were stained for human CD45. Gating on CD45+ cellsshowed human CD34+ CD38^(lo) cells in the bone marrow (first panel; BM);human CD19⁺ and human CD3+ lymphocytes in the spleen (second panel;spleen); and human CD3+ cells in the thymus (third panel; thymus). FIG.20C depicts a graphical representation of a FACS analysis of humansplenic B-cells labeled with CF SE and cultured in the presence ofmonoclonal antibodies to human CD40 and IgM. Human B-cells thatdeveloped in NSG xenochimaeric mice underwent proliferation followingstimulation of their antigen receptor. FIG. 20D depicts a graphicalrepresentation of the quantification of myeloerythroid colonies fromhuman CD34+CD38^(lo) cells obtained from the bone marrow of NSGxenochimaeric mice and plated on methycellulose. FIG. 20E depicts agraphical representation of the quantification of the development ofmyeloerythroid colonies following replating. FIG. 20F depict a graphicalrepresentation of the quantification of myeloid and lymphoid celldifferentiation (CD11b, CD33, CD3, and CD19 expression) in the CD45positive population of bone marrow cells expanded in vitro in a cocktailof cytokines (open circles) or a cocktail of cytokines supplemented withTat-Myc and Tat-Bcl-2 (black squares). FIG. 20G depicts a graphicalrepresentation of the quantification of myeloid and lymphoid celldifferentiation (CD11b, CD33, CD3, and CD19 expression) in the CD45positive population of spleen cells expanded in vitro in a cocktail ofcytokines (open circles) or a cocktail of cytokines supplemented withTat-Myc and Tat-Bcl-2 (black squares).

FIGS. 21A-21G depict a graphical representation of the expansion ofadult human G-CSF mobilized HSCs in vitro with Tat-Myc and Tat-Bcl-2.FIG. 21A depicts a graphical representation of the surface phenotype ofhuman CD45+ cells showing an enrichment of the human CD34+ and CD38+fraction. FIG. 21B depicts a graphical representation of the kinetics ofcell expansion in vitro over 18 days in culture in the presence ofTat-Myc and Tat Bcl-2. FIG. 21C depicts a graphical representationshowing that 5×10³ human adult G-CSF HSCs, expanded in vitro withTat-Myc and Tat-Bcl-2, gave rise to 4 morphologically distinct colonytypes in methylcellulose. FIG. 21D depicts a graphical representation ofFACS analysis showing that human adult G-CSF HSCs expanded in vitro withTat-Myc and Tat-Bcl-2 gave rise to human hematopoietic lineages inxenochimaeric NSG mice. Bone marrow was from NSG mice transplantedptlt-HSCs expanded with a cytokine cocktail supplemented with Tat-Mycand Tat-Bcl-2 (first panel; G-CSF+TMTB) or with fresh un-manipulatedcord blood cells (second panel; Fresh FCB). FIG. 21E depicts a graphicalrepresentation of FACS analysis of cells from bone marrow, spleen, andthymus. Bone marrow cells included human CD45 cells that were also humanCD34+ and CD38+(first panel), spleen cells included human CD45 cellsthat also stained for human CD3 (second panel), and thymus cellsincluded human CD45 cells as well as CD3 (third panel). FIG. 21F andFIG. 21G depict a graphical representation of a cohort of xenochimaericmice engrafted with 10⁶ G-CSF mobilized cells expanded in vitro in acocktail of cytokines supplemented with Tat-Myc and Tat-Bcl-2 (blacksquares) were assessed for myeloid and lymphoid cell differentiation.The CD45 positive population of bone marrow cells (FIG. 21F) and spleencells (FIG. 21G) were analyzed for CD11b, CD33, CD3, and CD19expression.

FIGS. 22A and 22B depict the activity of various Myc fusion proteinconstructs in an activated T cell viability assay. FIG. 22A depicts adiagrammatic alignment of some representative Myc fusion proteinconstructs. FIG. 22B depicts a graphical representation of the percentlive T cells 48 hours after treatment with representative Myc fusionprotein constructs.

FIGS. 23A-23C depict the activity of various Tat-fusion proteins (eachat 50 ug/ml) in an activated T cell viability assay. FIG. 23A depicts agraphical representation of the live gate from FACS analysis (forward Xside scatter) for untreated cells (No treatment). FIG. 23B depicts agraphical representation of the live gate from FACS analysis (forward Xside scatter) for Tat-Cre treated cells (Tat-Cre Control). FIG. 23Cdepicts a graphical representation of the live gate from FACS analysis(forward X side scatter) for Tat-Bcl2 treated cells (Tat-Bcl2). FIG. 23Ddepicts a graphical representation of the live gate from FACS analysis(forward X side scatter) for Tat-Myc treated cells (Tat-Myc).

FIG. 24 depicts the amino acid (SEQ ID NO: 1) and nucleic acid (SEQ IDNO: 2) sequences for some embodiments of the Tat-Myc polypeptide.

FIG. 25 depicts the amino acid (SEQ ID NO: 3) and nucleic acid (SEQ IDNO: 4) sequences for some embodiments of the Bcl-2 domain polypeptide.

FIGS. 26A and 26B depict primary HSCs also differentiate into mature redblood cells using differentiation media including Tat-Myc. FIG. 26Adepicts that on days 6 and 11, cells were assessed for GPAxCD71erythroid surface markers; and on day 11, the cells were also assessedfor adult and fetal hemoglobin expression by flow cytometry. FIG. 26Bdepicts that on day 10, a sample from the differentiation culture wascytospun on to a coverslip for H&E staining. Images are 10 and 20×magnification.

DETAILED DESCRIPTION

The present disclosure relates, among other things, to the in vitroproduction of red blood cells from hematopoietic stem cells (HSCs) byculturing HSCs with erythropoietin (EPO), optionally IL-3, and one ormore recombinant protein, or biologically active fragment thereof, thatpromotes one or more of cell survival or proliferation. Thedifferentiation process described herein does not require feeder cells(e.g., fibroblasts) and/or serum, and results in the production ofmature anucleated red blood cells in about 7 to 14 days.

The methods of producing mature red blood cells described herein can beused with HSCs from any source, including but not limited to, bonemarrow, peripheral blood, mobilized peripheral blood, cord blood, andplacenta, as well as HSCs produced from embryonic stem cells and inducedpluripotent stem cells. HSCs isolated from any source may be used toproduce, without limitation, universal donor red blood cells, red bloodcells of a rare blood type, red blood cells for personalized medicine(e.g., autologous transfusion, optionally with pay-loading or geneticengineering), and red blood cells engineered to include one or moreproteins of interest.

Production of mature red blood cells from HSCs in vitro, using themethods of the present disclosure, can be enhanced by usingconditionally immortalized HSCs. Enhancement may include, but is notlimited to, one or more of increased numbers of mature red blood cellsper starting HSC, improvement of one or more characteristics of thepopulation of mature red blood cells, or decreased numbers of days togenerate mature red blood cells. Conditionally immortalized HSCs may beproduced by exposing HSCs to a first protein that promotes cellproliferation and/or cell survival and a second protein that inhibitsapoptosis, using one or more of any transgenic approach and/or proteintransduction approach known in the art.

The present disclosure also relates, among other things, to populationsof mature red blood cells produced by one or more of the methods of thepresent disclosure. Populations of mature red blood cells may becharacterized by one or more characteristics, including but not limitedto, at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, or 100% of the cells in the populationbeing anucleated cells, expressing increased levels of GPA, increasedlevels of adult hemoglobin (second decade or higher by FACS), decreasedlevels of CD71 (e.g. GPA⁺/CD71⁻) expression, and decreased levels offetal hemoglobin (first decade; 0-10 by FACS). Populations of mature redblood cells may also include one or more recombinant proteins ofinterest. These proteins of interest may be useful in prevention,treatment, or diagnosis of one or more diseases or disorders.

The present disclosure also relates, among other things, topharmaceutical compositions including populations of mature red bloodcells produced by one or more of the methods described herein. Thesepharmaceutical compositions may also include one or more exogenousproteins of interest useful in prevention, treatment, or diagnosis ofone or more diseases or disorders.

The present disclosure also relates, among other things, to methods oftreating, preventing, or diagnosing a disease or a disordercharacterized by a deficiency of red blood cells by providing to asubject in need thereof a population of mature red blood cells of thepresent disclosure. The present disclosure also relates, among otherthings, to methods of treating or preventing a disease or a disorder byadministering one or more population of red blood cells orpharmaceutical composition of the present disclosure that includes oneor more proteins of interest to a subject, where the one or moreproteins of interest are useful in treating or preventing the disease ordisorder.

The present disclosure also relates, among other things, to methods forextending the life or half-life of red blood cells or population of redblood cells in vitro. Extension of the red blood cell half life in vitroexpands the ability of blood banks to store and supply blood forpatients (e.g., civilian and armed forces) in need. As described herein,maintenance of red blood cells in media containing one or more exogenouspolypeptide that inhibits apoptosis increases the half-life ofpopulation of red blood cells in vitro.

Proteins of the Present Disclosure

Certain aspects of the present disclosure relate to the in vitroproduction of a population of red blood cells from hematopoietic stemcells (HSCs), maintenance of a population of red blood cells, byculturing the HSCs and/or red blood cells in the presence of mediacontaining one or more recombinant proteins (such as exogenousproteins), or biologically active fragment thereof, that promote cellsurvival and/or proliferation and/or inhibit apoptosis. Further aspectsof the present disclosure relate to HSCs and/or red blood cells of thepresent disclosure that contain, include, and/or express one or moreproteins of interest.

As used herein, a protein that “promotes cells survival and/orproliferation” refers to a protein whose biological activity eitherdirectly or indirectly activates, induces, enhances, stimulates, allows,or increases cell survival and/or cell proliferation. As used herein, a“biologically active fragment” of a protein of the present disclosure,such as a protein that promotes cell survival and/or proliferation or aprotein that inhibits apoptosis, is a fragment of a full-length proteinof the present disclosure that retains at least one activity and/orfunction of the full-length protein.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably and refer to a polymer of amino acid residues. Theterms apply to naturally occurring amino acid polymers as well as aminoacid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analogue. Asused herein, the terms encompass amino acid chains of any length,including full length proteins, where the amino acid residues are linkedby covalent peptide bonds

Recombinant Proteins that Promote Cell Survival and/or CellProliferation

Certain aspects of the present disclosure relate to the in vitroproduction of a population of red blood cells from hematopoietic stemcells (HSCs) by culturing the HSCs in the presence of media containingone or more recombinant protein, or biologically active fragmentthereof, that promotes cell survival and/or proliferation.

Any suitable protein known in the art that promotes cell survival and/orproliferation may be used. In some embodiments, the one or morerecombinant protein that promotes cell survival and/or proliferation isan onco-peptide (e.g., a polypeptide encoded by a proto-oncogene and/oroncogene). Suitable onco-peptides may be of any suitable class thatinduces cell immortality. Examples of suitable onco-peptides that thatpromotes cell survival and/or proliferation include, without limitation,growth factors and/or mitogens (e.g., PDGF-derived growth factors suchas c-Sis); receptor tyrosine kinases, particularly constitutively activereceptor tyrosine kinases (e.g., epidermal growth factor receptor(EGFR), thrombocyte-derived growth factor receptor (PDGFR), vascularendothelial growth factor receptor (VEGFR), and HER2/neu); cytoplasmictyrosine kinases (e.g., Src-family, Syk-ZAP-70 family, and BTK family oftyrosine kinases); cytoplasmic serine/threonine kinases and theirregulatory subunits (e.g., Raf kinases, cyclin-dependent kinases,members of the Akt family); regulatory GTPases (e.g., Ras protein);transcription factors (e.g., MYC and HIF-1a); telomerase reversetranscriptases (e.g., TERT or hTERT); and/or factors that activate otheronco-peptides (e.g., cyclins, including cyclins A, B, D, and/or E, suchas cyclin D1 and D3).

In certain embodiments, a protein that promotes cell survival and/orproliferation is, by way of non-limiting example, MYC, mTOR, cyclin D1,cyclin D3, STAT3, STAT5, AML-ETO, AKT, ICN-1, hTERT, PDK-1, MLL-ENL, IL3receptor .beta. chain, .beta.-catenin, Hedgehog family (Shh, Ihh, Dhh),Bmi-1, c-Jun, Wnt, Bcl-2, Bcl-6, Bcl-10, epidermal growth factorreceptor (EGFR, ErbB-1, HER1), ErbB-2 (HER2/neu), ErbB-3/HER3,ErbB-4/HER4, EGFR ligand family; insulin-like growth factor receptor(IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family;platelet derived growth factor receptor (PDGFR) family, PDGFR ligandfamily; fibroblast growth factor receptor (FGFR) family, FGFR ligandfamily, vascular endothelial growth factor receptor (VEGFR) family, VEGFfamily; HGF receptor family; TRK receptor family; ephrin (EPH) receptorfamily; AXL receptor family; leukocyte tyrosine kinase (LTK) receptorfamily; TIE receptor family, angiopoietin 1,2; receptor tyrosinekinase-like orphan receptor (ROR) receptor family; discoidin domainreceptor (DDR) family; RET receptor family; KLG receptor family; RYKreceptor family; MuSK receptor family; Transforming growth factor .beta.(TGF-.beta.) receptors, TGF-.beta.; Cytokine receptors, Class I(hematopoietin family) and Class II (interferon/IL-10 family) receptors,tumor necrosis factor (TNF) receptor superfamily (TNFRSF), deathreceptor family; cancer-testis (CT) antigens, lineage-specific antigens,differentiation antigens, alpha-actinin-4, ARTC1, breakpoint clusterregion-Abelson (Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5),caspase-8 (CASP-8), .beta.-catenin (CTNNB1), cell division cycle 27(CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusionprotein, EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acutemyeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin(FN), GPNMB, low density lipid receptor/GDP-L fucose: .beta.-Dgalactose2-a-Lfucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine toisoleucine exchange at residue 170 of the .alpha.-helix of the.alpha.2-domain in the HLA-A2 gene (HLA-A*201-R170I), HLA-A11, heatshock protein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanomaubiquitous mutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase(PAP), neo-PAP, Myosin class I, NFYC, OGT, OS-9, pml-RARalpha fusionprotein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600,SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TriosephosphateIsomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8,GnT-V (aberrant N-acetyl glucosaminyl transferase V, MGAT5), HERV-K-MEL,KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma(CAMEL), MAGE-A 1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1,MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1),MART-1/Melan-A (MLANA), gp100, gp100/Pmell7 (SILV), tyrosinase (TYR),TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17, SSX-1,2,3,4,TRP2-INT2, carcino-embryonic antigen (CEA), Kallikrein 4, mammaglobin-A,OA1, prostate specific antigen (PSA), TRP-1/gp75, TRP-2, adipophilin,interferon inducible protein absent in melanoma 2 (AIM-2), BING-4, CPSF,cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblastgrowth factor-5 (FGF-5), glycoprotein 250 (gp250), EGFR (ERBB1),HER-2/neu (ERBB2), interleukin 13 receptor .alpha.2 chain (ILI3Ralpha2), IL-6 receptor, intestinal carboxyl esterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUC1, p53 (TP53), PBF, PRAME, PSMA, RAGE-1,RNF43, RU2AS, SOX10, STEAP1, survivin (BIRC5), human telomerase reversetranscriptase (hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT,SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS,HOM-TES-85, AF15q14, HCA661, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35,FTHL17, NXF2, TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotypes,Bence-Jones protein, estrogen receptors (ER), androgen receptors (AR),CD40, CD30, CD20, CD19, CD33, cancer antigen 72-4 (CA 72-4), cancerantigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen125 (CA 125), cancer antigen 19-9 (CA 19-9), β-human chorionicgonadotropin, squamous cell carcinoma antigen, neuron-specific enolase,heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline(707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4),carcinoembryogenic antigen peptide-1 (CAP-1), calcium-activated chloridechannel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2(HST-2), simian virus 40 (SV40) derived transforming genes and proteins,Human papilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minorcapsid antigens, others), Epstein-Ban virus (EBV) proteins (EBV latentmembrane proteins—LMP1, LMP2; others), Hepatitis B or C virus proteins,Human immunodeficiency virus (HIV) proteins, functional homologues,functional analogues, or biologically active fragments thereof.

In some embodiments, a protein that promotes cell survival and/orproliferation is a protein that inhibits an endogenous antagonist (e.g.,protein and/or gene) of cell survival and/or proliferation. For example,the protein may be an inhibitor of a transcriptional repressor thatsuppresses expression of a gene that promotes cell survival and/orproliferation. In certain embodiments, the transcriptional repressor mayantagonize an onco-peptide of the present disclosure that regulatesexpression of a gene that promotes cell survival and/or proliferation,such as MYC. For example, in some embodiments, the protein inhibits atleast one member of the MAD family of transcriptional repressors, e.g.,MAD-1; or cyclin-dependent kinase inhibitors (e.g., p16, p19, p21, orp27).

In other embodiments, cell survival and/or proliferation is promoted byan agent that inhibits an endogenous antagonist (e.g., protein and/orgene) of cell survival and/or proliferation. For example, the agent maybe a genetic inhibitor or a small molecule inhibitor (such as, anantagonist). In some embodiments, the agent may include an inhibitor ofa transcriptional repressor that suppresses expression of a gene thatpromotes cell survival and/or proliferation. In certain embodiments, thetranscriptional repressor antagonizes an onco-peptide of the presentdisclosure that regulates expression of a gene that promotes cellsurvival and/or proliferation, e.g., MYC. For example, in someembodiments, the agent that inhibits at least one member of the MADfamily of transcriptional repressors, e.g., MAD-1. In certain otherembodiments, the agent is an inhibitor of cyclin-dependent kinaseinhibitors (e.g., p16, p19, p21, or p27).

Any agent that inhibits an endogenous antagonist of a protein thatpromotes cell survival and/or proliferation relative to a wild-type cellof the same type is suitable for use in the methods of the presentdisclosure. An agent that is an inhibitor of an endogenous antagonist ofa protein that promotes cell survival and/or proliferation reduces,inhibits, or decreases the activity or level of the antagonist at anystage or by any mechanism. For example, in some instances, such an agentinterferes with expression of an agent that antagonizes the activity ofa protein that promotes cell survival and/or proliferation, e.g., at thetranslational level or at the transcriptional level. In certainembodiments, an agent that interferes with expression of an agent thatantagonizes the activity of a protein that promotes cell survival and/orproliferation is an agent capable of RNA interference (an RNAimolecule). In some embodiments, an RNAi molecule is generated bycleavage of or binding to mRNA encoding a polypeptide. An RNAi moleculeis generated by any suitable means, including by small interfering RNA(siRNA), microRNA (miRNA), double stranded RNA (dsRNA), or small hairpinRNA (shRNA). In certain embodiments, an agent that interferes withexpression of an agent that antagonizes the activity of a protein thatpromotes cell survival and/or proliferation is a small molecule, e.g., asmall organic molecule.

In other embodiments, the agent inhibits the activity or level of aprotein that anatgonizes cell survival and/or proliferation. In someembodiments, the agent acts directly on the protein that anatgonizescell survival and/or proliferation. For example, the agent may bind toand inhibit the activity of the protein that anatgonizes cell survivaland/or proliferation. Accordingly, in certain embodiments the agent isan antibody or small molecule that binds to and disrupts the naturalfunction of a protein that anatgonizes cell survival and/orproliferation.

In other embodiments, the protein that promotes cell survival and/orproliferation further includes a protein transduction domain (PTD).

In some embodiments, protein that promotes cell survival and/orproliferation is provided as a bolus. As used herein, a “bolus” refersto an amount or concentration of a protein that is given to a subjectthe increase the concentration of the protein in the blood of thesubject to an effective level. A bolus may be administered by anysuitable method known in the art. In some embodiments, the bolus isprovided about every 12 hours, about every 24 hours, about every 36hours, about every 48 hours, about every 60 hours, or about every 72hours.

In some embodiments, the one or more recombinant protein that promotescell survival and/or cell proliferation is a MYC polypeptide, ahomologue thereof or a biologically active fragment thereof. In someembodiments, the one or more recombinant protein that promotes cellsurvival and/or cell proliferation is an ICN-1 polypeptide, a homologuethereof or a biologically active fragment thereof. In certainembodiments, the one or more recombinant protein that promotes cellsurvival and/or cell proliferation is a PTD-MYC fusion protein. Incertain embodiments, the one or more recombinant protein that promotescell survival and/or cell proliferation is a PTD-ICN-1 fusion protein.

MYC

A MYC polypeptide of the present disclosure includes, withoutlimitation, any polypeptide, or fragment thereof, having the activity ofa MYC protein.

As used herein, “MYC” and “MYC protein” are used interchangeably andrefer to a protein that is a member of the MYC family of bHLH (basichelix-loop-helix) transcription factors. MYC proteins of the presentdisclosure are transcription factors that regulate expression of MYCresponsive genes, and as such enter the nucleus of a cell to function.MYC activity can activate expression of certain MYC responsive genes,while repressing expression of other MYC responsive genes. MYC activitycan regulate various cellular functions including, without limitation,cell proliferation, cell growth, cell survival, and apoptosis.

As described herein, transient expression of MYC, provided from anexogenous or endogenous source, during red blood cell production fromHSCs can increase the yield of mature red blood cells, can decrease thelength of time for the production of mature red blood cells, canincrease the percent of mature red blood cells in a population, and/orcan increase the rate of production of mature red blood cells ascompared to production in the absence of MYC. Without wishing to bebound by theory, it is believed that it is the transient expression oflow levels of MYC that promote HSC differentiation to mature red bloodcells, as it has been shown that prolonged expression of MYC inembryonic stem cells promotes self-renewal by inhibiting differentiation(e.g., Cartwright et al., Development. 2005 March; 132(5):885-96).Moreover, the ability of transient expression of MYC at low levels toenhance the production of mature red blood cells from HSCs is surprisinggiven the recent finding that ectopic high levels of MYC expressioninhibited differentiation of erythroid progenitor cells to anucleatedred blood cells (Jayapal et al., J Biol Chem. 2010 Dec. 17;285(51):40252-65).

PTD-MYC fusion proteins of the present disclosure allow for an increasein MYC activity in HSCs by the exogenous addition of MYC, without theneed for overexpressing endogenous MYC in the HSCs or recombinantlyexpressing MYC via genetic manipulation of the HSCs.

MYC polypeptides of the present disclosure include, without limitation,full length MYC proteins, fragments that retain the activity of afull-length MYC protein, homologues thereof, and analogues thereof. MYCpolypeptides of the present disclosure may be produced by any suitablemethod known in the art. For example, a MYC polypeptide may be purifiedfrom a native source, may be recombinantly expressed, or may bechemically synthesized.

MYC Proteins

Examples of full length MYC proteins suitable for use in any of themethods of the present disclosure include, without limitation, c-Myc,N-Myc, L-Myc, v-MYC, and S-Myc.

In certain preferred embodiments, the MYC polypeptide is a full-lengthc-Myc polypeptide. The c-Myc polypeptide may have one or more of thefollowing features: the polypeptide may be a polymer of 439 amino acids,the polypeptide may have a molecular weight of 48.804 kDa, thepolypeptide may contain a basic Helix-Loop-Helix Leucine Zip-per(bHLH/LZ) domain, or the polypeptide may bind to a sequence containingCACGTG (i.e., an E-box sequence). Preferably, the c-Myc polypeptide isthe human c-Myc polypeptide having NCBI Accession Number NP_002458.2.Moreover, a c-Myc polypeptide of the present disclosure may be a c-Mycpolypeptide that has not undergone any post-translational modifications.Alternatively, a c-Myc polypeptide of the present disclosure may be ac-Myc polypeptide that has undergone post-translational modifications.

Biologically Active MYC Fragments

In other embodiments, a MYC polypeptide of the present disclosure is abiologically active fragment of a full-length MYC protein that retainsat least one activity of a full-length MYC protein. The MYC polypeptidemay be a fragment of c-Myc, N-Myc, L-Myc, or S-Myc. A MYC fragment ofthe present disclosure may contain at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 350, atleast 400, or more consecutive amino acid residues of the amino acidsequence of a MYC protein.

MYC Homologues and Analogues

In other embodiments, a MYC polypeptide of the present disclosure is ahomologue or analogue of a MYC protein, or a fragment thereof, thatretains at least one activity of a full-length MYC protein.

For example, a MYC polypeptide of the present disclosure may include anamino acid sequence that is at least 40% to 100% identical, e.g., atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about40% to about 100% identical to a MYC protein or fragments thereof. Incertain embodiments, the MYC polypeptide is a homologue or an analogueof c-Myc, N-Myc, L-Myc, S-Myc, or fragments thereof.

MYC polypeptides of the present disclosure also include functionalhomologues or analogues of the human c-Myc polypeptide having NCBIAccession Number NP_002458.2, or a fragment thereof. In certainembodiments, the c-Myc homologue or analogue contains an amino acidsequence that is at least 40% to 100% identical, e.g., at least 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 40% toabout 100% identical to the c-Myc polypeptide sequence of NCBI AccessionNumber NP_002458.2 or fragment thereof.

In other embodiments, the c-Myc homologue or analogue contains apolypeptide sequence of at least 10 amino acids, at least 20 aminoacids, at least 30 amino acids, at least 40 amino acids, at least 50amino acids, at least 60 amino acids, at least 70 amino acids, at least80 amino acids, at least 90 amino acids, at least 100 amino acids, atleast 150 amino acids, at least 200 amino acids, at least 250 aminoacids, at least 300 amino acids, at least 350 amino acids, at least 400amino acids, or more in length that is at least 50% to 100% identical,e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about50% to about 100% identical to the c-Myc polypeptide sequence of NCBIAccession Number NP_002458.2 or fragment thereof.

As used herein, a “homologue” refers to a protein or polypeptide havingamino acid sequence similarity between a reference sequence and at leasta fragment of a second sequence. Homologues may be identified by anymethod known in the art, preferably, by using the BLAST tool to comparea reference sequence to a single second sequence or fragment of asequence or to a database of sequences. As described below, BLAST willcompare sequences based upon percent identity and similarity.

The terms “identical” or percent “identity,” in the context of two ormore sequences (e.g., amino acid sequences), refer to two or moresequences or subsequences that are the same. Two sequences aresubstantially identical if two sequences have a specified percentage ofamino acid residues or nucleotides that are the same (i.e., 29%identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or 100% identity over a specified region, or, whennot specified, over the entire sequence), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the identity existsover a region that is at least about 10 amino acids in length, or morepreferably over a region that is 20, 50, 200, or more amino acids inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastp, the default parameters are Gap opening penalty=11and Gap extension penalty=1. For blastn, the default parameters are Gapopening penalty=5 and Gap extension penalty=2.

As used herein, a “comparison window” includes reference to a segment ofany one of the number of contiguous positions including, but not limitedto from 20 to 600, usually about 50 to about 200, more usually about 100to about 150 in which a sequence may be compared to a reference sequenceof the same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman (1981), by the homology alignment algorithm of Needleman andWunsch (1970) J Mol Biol 48(3):443-453, by the search for similaritymethod of Pearson and Lipman (1988) Proc Natl Acad Sci USA85(8):2444-2448, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection [see, e.g., Brent et al., (2003)Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (RingbouEd)].

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nucleic AcidsRes 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol215(3)-403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. For aminoacid sequences, the BLASTP program uses as defaults a wordlength of 3,and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoffand Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919]alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. For nucleotide sequences, the BLASTN program uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4,and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Other than percentage of sequence identity noted above, anotherindication that two polypeptides are substantially identical is that thefirst polypeptide is immunologically cross-reactive with antibodiesraised against the second polypeptide. Thus, a polypeptide is typicallysubstantially identical to a second polypeptide, for example, where thetwo peptides differ only by conservative substitutions.

As disclosed herein, suitable MYC polypeptides also includeconservatively modified variants of MYC polypeptides of the presentdisclosure. “Conservatively modified variants” as used herein includeindividual substitutions, deletions, or additions to an encoded aminoacid sequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.The following eight groups contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)).

Proteins Downstream of MYC

In other embodiments, a suitable protein of the present disclosure thatpromotes cell survival and/or proliferation is a protein that promotescell survival and/or proliferation and that is downstream of MYC in aMYC pathway. Any protein downstream known in the art is suitable for usewith the methods of the present disclosure. Examples of suitableproteins that promote cell survival and/or proliferation and that aredownstream of MYC include, without limitation, AKT and AKT-relatedproteins, such as PDK-1, mTORC2, PI3K-delta. The protein downstream ofMYC that promotes cell survival and/or proliferation may further be aPTD-fusion protein. Accordingly, in certain embodiments, the proteindownstream of MYC that promotes cell survival and/or proliferation is anAKT-PTD fusion protein, a PTD-PDK-1 fusion protein, a PTD-mTORC2 fusionprotein, or a PTD-PI3K-delta fusion protein.

In other embodiments, cell survival and/or proliferation is promoted inHSCs of the present disclosure by inhibiting a protein that antagonizecell survival and/or proliferation and that is downstream of MYC in aMYC pathway. Examples of proteins that antagonize cell survival and/orproliferation and that are downstream of MYC include, withoutlimitation, of pTEN, PP2A, PHLPP, CTMP. Accordingly, in certainembodiments, cell survival and/or proliferation is promoted in HSCs ofthe present disclosure by inhibiting pTEN, PP2A, PHLPP, and/or CTMP. Anymethod known in the art for inhibiting protein and/or gene expression,activity, and/or function may be used, including without limitation themethods disclosed herein. Non-limiting examples include geneticinhibitors, small molecule inhibitors, RNA interference, and antibodies.

Activities of Full-Length MYC Proteins

In other embodiments, a MYC protein or PTD-MYC fusion protein of thepresent disclosure contains a full-length MYC polypeptide having atleast one MYC activity, a fragment of a MYC protein that retains atleast one activity of a full-length MYC protein, a homologue of a MYCprotein that retains at least one activity of a full-length MYC protein,or an analogue of a MYC protein that retains at least one activity of afull-length MYC protein.

Full-length MYC proteins of the present disclosure have numerousactivities. Examples of such activities include, without limitation,transcription factor activity, protein binding activity, nucleic acidbinding activity, cell proliferation regulation activity, cell growthregulation activity, apoptosis regulation activity, morphogenesisregulation activity, development regulation activity, and enhancedhematopoietic compartment reconstitution activity.

In some embodiments, a MYC protein or PTD-MYC fusion protein of thepresent disclosure has a MYC activity that together with EPO and,optionally, IL-3 produce mature red blood cells from HSCs. In otherembodiments, a MYC protein or PTD-MYC fusion protein of the presentdisclosure has a MYC activity that conditionally immortalizes HSCs.Advantageously, administering MYC in the form of a PTD-MYC fusionprotein results in transient MYC activity in HSCs. In some embodiments,the level of transient MYC activity is sufficient to enhance HSCdifferentiation to mature red blood cells. Additionally, a PTD-MYCfusion protein of the present disclosure can increase the intracellularlevels of MYC in HSCs, which results in an expansion of the HSCs. Thistransient MYC activity avoids the potentially negative effects ofprolonged MYC activity in the cells, such as uncontrolled cell growthand oncogenic transformation. Moreover, the use of a PTD-MYC fusionprotein allows for the induction of MYC activity in the HSCs withoutgenetically modifying the cells.

Exogenous Proteins that Inhibit Apoptosis

Certain aspects of the present disclosure relate to the in vitromaintenance of red blood cells or a population of red blood cells of thepresent disclosure by culturing the red blood cells or population of redblood cells in the presence of media including one or more recombinantprotein (such as an exogenous protein) that inhibits apoptosis. Otheraspects of the present disclosure also relate to further culturing HSCsof the present disclosure in the presence of media including one or moreexogenous protein that inhibits apoptosis. As used herein a protein orpolypeptide that “inhibits apoptosis” refers to a protein or polypeptidewhose function either directly or indirectly reduces, prevents, ordecreases a process associated with apoptosis (i.e., programmed celldeath).

Any suitable protein known in the art that inhibits apoptosis may beused. In some embodiments, the protein that inhibits apoptosis is aprotein that contains one or more Bcl-2 homology domains. Examples ofproteins that contain one or more Bcl-2 homology domains include,without limitation, Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, A1, Bfl-1, andBcl-w.

In some embodiments, a protein that inhibits apoptosis is a protein thatinhibits any endogenous protein and/or gene known in the art thatpromotes apoptosis. Examples of proteins that promote apoptosis include,without limitation, Bcl-2 family members, caspases, and proteins of theTNF family of receptors.

In other embodiments, inhibition of apoptosis is achieved by an agentthat inhibits an endogenous protein and/or gene known in the art thatpromotes apoptosis. For example, the agent may be a genetic inhibitor ora small molecule inhibitor (such as, an antagonist). Any geneticinhibitor or small molecule inhibitor known in the art for inhibitingapoptosis may be used. In some embodiments, the agent interferes withexpression of a protein that promotes apoptosis. For example, the agentmay interfere with expression a protein that promotes apoptosis at thetranslational level or at the transcriptional level. In someembodiments, RNA interference is used to interfere with the expressionof a protein that promotes apoptosis. In other embodiments, the agentinhibits the activity or level of a protein that promote apoptosis. Insome embodiments, the agent acts directly on the protein that promotesapoptosis. For example, the agent may bind to and inhibit the activityof a protein that promotes apoptosis. Accordingly, in certainembodiments the agent is an antibody or small molecule that binds to anddisrupts the natural function of a protein that inhibits apoptosis.

In some embodiments, the one or more exogenous protein that inhibitsapoptosis is one or more protein that contains one or more Bcl-2homology domains. In some embodiments, the one or more exogenous proteinthat inhibits apoptosis further includes a protein transduction domain(PTD). In some embodiments, the one or more exogenous protein thatinhibits apoptosis is a PTD-Bcl-2 fusion protein.

Bcl-2

A Bcl-2 polypeptide of the present disclosure includes, withoutlimitation, any polypeptide, or fragment thereof, having the activity ofa Bcl-2 protein.

As used herein, “Bcl-2,” “Bcl-2 polypeptide,” and “Bcl-2 protein” areused interchangeably and refer to a protein that is a member of theBcl-2 protein family that has one or more and/or all Bcl-2 homology (BH)domains, such as but not limited to, BH1, BH2, BH3, and BH4. Members ofthe bcl-2 protein family typically form heterodimer or homodimers, andfunction as regulators of apoptosis. In certain preferred embodiments,Bcl-2 polypeptides of the present disclosure have anti-apoptoticactivity and/or an activity useful in the process of conditionalimmortalization of HSCs.

As described herein, the addition of exogenous Bcl-2 to a population ofred blood cells can increase the length of time the red blood cellsremain viable in vitro, can increase the percentage of red blood cellsthat remain viable over time, and/or can delay and/or reduce the loss ofone or more red blood cell functional characteristics over time invitro, as compared with a corresponding population of red blood cellsthat is maintained in the absence of Bcl-2. In some embodiments, theaddition of exogenous Bcl-2 is to red blood cell storage media. The oneor more functional characteristics may include, but are not limited to,one or more of oxygen carrying capacity, amount of hemoglobin, type ofhemoglobin expressed (adult vs. fetal), level of transferrin receptorexpressed on the surface, or one or more mature red blood cell markers.

In another embodiment, transient up-regulation of Bcl-2, provided froman exogenous or endogenous source during the final stages of red bloodcell production from HSCs, may increase the length of time the red bloodcells remain viable in vitro, may increase the percentage of red bloodcells that remain viable over time, and/or may delay and/or reduce theloss of one or more red blood cell functional characteristics over timein vitro, as compared with maintenance in the absence of Bcl-2. In someembodiments, the transient up-regulation of Bcl-2, provided from anexogenous or endogenous source during the final stages of red blood cellproduction from HSCs, is designed to enhance RBC maintenance in redblood cell storage media.

In another embodiment, the present disclosure is drawn to red blood cellstorage media containing Bcl-2.

PTD-Bcl-2 fusion proteins of the present disclosure allow for anincrease in Bcl-2 activity in HSCs by the exogenous addition of Bcl-2,without the need for overexpressing endogenous Bcl-2 in the HSCs orrecombinantly expressing Bcl-2 via genetic manipulation of the HSCs.

Bcl-2 polypeptides of the present disclosure include, withoutlimitation, full length Bcl-2 proteins, fragments that retain theactivity of a full-length Bcl-2 protein, homologues thereof, andanalogues thereof. In some embodiments, Bcl-2 fragments that retain theactivity of a full-length Bcl-2 protein include a truncated form ofBcl-2 that has been deleted for the unstructured loop domain (Anderson,M., et al. (1999). Refolding, purification and characterization of aloop deletion mutant of human Bcl-2 from bacterial inclusion bodies.Prot Expr. Purif. 15, 162-70). Bcl-2 polypeptides of the presentdisclosure may be produced by any suitable method known in the art. Forexample, a Bcl-2 polypeptide may be purified from a native source, maybe recombinantly expressed, or may be chemically synthesized.

Bcl-2 Proteins

Examples of full length Bcl-2 proteins suitable for use in any of themethods of the present disclosure include, without limitation, Bcl-2,Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1, Bfl-1, and Bcl-w.

In certain preferred embodiments, the Bcl-2 polypeptide is a full-lengthhuman Bcl-2 polypeptide that has been deleted for the unstructured loopdomain. The human Bcl-2 polypeptide may have one or more of thefollowing features: the polypeptide may be a polymer of 239 amino acids,the polypeptide may have a molecular weight of approximately 26.3 kDa,or the polypeptide may contain at least one Bcl-2 homology (BH) domain,such as BH1, BH2, BH3, and BH4. Preferably, the human Bcl-2 polypeptideis the Bcl-2 polypeptide having NCBI Accession Number NP_000624.2.Moreover, a Bcl-2 polypeptide of the present disclosure may be a Bcl-2polypeptide that has not undergone any post-translational modifications.Alternatively, a Bcl-2 polypeptide of the present disclosure may be aBcl-2 polypeptide that has undergone post-translational modifications.

Biologically Active Bcl-2 Fragments

In other embodiments, a Bcl-2 polypeptide of the present disclosure is abiologically active fragment of a full-length Bcl-2 protein that retainsat least one activity of a full-length Bcl-2 protein. The Bcl-2polypeptide may be a fragment of Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9,Bcl-2 related protein A1, Bfl-1, or Bcl-w.

A Bcl-2 fragment of the present disclosure may contain at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, at least 100, at least 110, at least 120, at least 130, at least140, at least 150, at least 160, at least 170, at least 180, at least190, at least 200, at least 210, at least 220, at least 230, or moreconsecutive amino acid residues of the amino acid sequence of a Bcl-2protein.

Bcl-2 Homologues and Analogues

In other embodiments, a Bcl-2 polypeptide of the present disclosure is ahomologue or analogue of a Bcl-2 protein or fragment thereof. Forexample, a Bcl-2 polypeptide of the present disclosure may include anamino acid sequence that is at least 40% to 100% identical, e.g., atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about40% to about 100% identical to a Bcl-2 protein or fragments thereof. Incertain embodiments, the Bcl-2 polypeptide is a homologue or analogue ofBcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1, Bfl-1,Bcl-w, or fragments thereof.

Bcl-2 polypeptides of the present disclosure also include functionalhomologues or analogues of the human Bcl-2 polypeptide having NCBIAccession Number NP_00624.2, or a fragment thereof. In certainembodiments, the Bcl-2 homologue or analogue contains an amino acidsequence that is at least 40% to 100% identical, e.g., at least 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 40% toabout 100% identical to the Bcl-2 polypeptide sequence of NCBI AccessionNumber NP_00624.2 or fragment thereof.

In other embodiments, the Bcl-2 homologue or analogue contains apolypeptide sequence of at least 10 amino acids, at least 20 aminoacids, at least 30 amino acids, at least 40 amino acids, at least 50amino acids, at least 60 amino acids, at least 70 amino acids, at least80 amino acids, at least 90 amino acids, at least 100 amino acids, atleast 110 amino acids, at least 120 amino acids, at least 130 aminoacids, at least 140 amino acids, at least 150 amino acids, at least 160amino acids, at least 170 amino acids, at least 180 amino acids, atleast 190 amino acids, at least 200 amino acids, at least 210 aminoacids, at least 220 amino acids, at least 230 amino acids, or more inlength that is at least 50% to 100% identical, e.g., at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%,96%, 97%, 98%, or any other percent from about 50% to about 100%identical to the Bcl-2 polypeptide sequence of NCBI Accession NumberNP_00624.2 or fragment thereof.

As disclosed herein, suitable Bcl-2 polypeptides also includeconservatively modified variants of Bcl-2 polypeptides of the presentdisclosure.

Bcl-2 Homologues that Promote Apoptosis

In some embodiments, apoptosis is inhibited by inhibiting a protein thatcontains one or more BH domains and that promotes apoptosis. Examples ofBH domain-containing proteins that promote apoptosis include, withoutlimitation, Bcl-Xs, BIM, PUMA, NOXA, NOXA-2, DIVA, BAK, BAX, BIK, BAD,BID, and EGL-1. Accordingly, in certain embodiments, apoptosis isinhibited by inhibiting Bcl-Xs, BIM, PUMA, NOXA, NOXA-2, DIVA, BAK, BAX,BIK, BAD, BID, and/or EGL-1. Any method known in the art for inhibitingprotein and/or gene expression, activity, and/or function may be used,including without limitation the methods disclosed herein. Non-limitingexamples include genetic inhibitors, small molecule inhibitors, RNAinterference, and antibodies.

Activities of Full-Length Bcl-2 Proteins

In other embodiments, a Bcl-2 protein or a PTD-Bcl-2 fusion protein ofthe present disclosure contains a full-length Bcl-2 polypeptide havingat least one Bcl-2 activity, a fragment of a Bcl-2 protein that retainsat least one activity of a full-length Bcl-2 protein, a homologue of aBcl-2 protein that retains at least one activity of a full-length Bcl-2protein, or an analogue of a Bcl-2 protein that retains at least oneactivity of a full-length Bcl-2 protein.

Full-length Bcl-2 proteins of the present disclosure have numerousactivities. Examples of such activities include, without limitation,apoptosis regulation activity, cell survival regulation activity,protein binding activity, mitochondrial membrane permeability regulationactivity, caspase regulation activity, voltage-dependent anion channelregulation activity, G2 checkpoint regulation activity, outermitochondrial membrane channel (VDAC) regulation activity, mitochondrialmembrane potential regulation activity, protein channel activity, andcytochrome C regulation activity.

In some embodiments, a Bcl-2 protein or a PTD-Bcl-2 fusion protein ofthe present disclosure has a Bcl-2 activity that aids in the productionof mature red blood cells from HSCs, in the maintenance of red bloodcells or a population of red blood cells, and/or in the immortalizationof HSCs. Advantageously, administering Bcl-2 in the form of a PTD-Bcl-2fusion protein results in transient Bcl-2 activity in HSCs. Thistransient Bcl-2 activity avoids any potentially negative effects ofprolonged Bcl-2 activity in the cells. Moreover, the use of a PTD-Bcl-2fusion protein allows for the induction of Bcl-2 activity in the redblood cells and/or HSCs without genetically modifying the cells, andallows for maintenance of anucleated mature red blood cells in vitro.

Proteins of Interest

Certain aspects of the present disclosure relate to the in vitroproduction of a population of red blood cells from HSCs; the in vitromaintenance of red blood cells or a population of red blood cells,and/or the administration of red blood cells to a subject in needthereof, where the red blood cells or population of red blood cellsincludes one or more proteins of interest. Aspects of the presentdisclosure also include population of red blood cells including one ormore proteins of interest, and pharmaceutical compositions includingsuch population of red blood cells. In some embodiments, the one or moreprotein of interest further includes a protein transduction domain(PTD). In some embodiments, the protein of interest is a PTD-protein ofinterest fusion protein.

The proteins of interest may include, but are not limited to, one ormore growth factors, hormones, or other polypeptides useful forprevention, diagnosis and/or treatment of one or more diseases ordisorders. Use of red blood cells or populations of red blood cells ofthe present disclosure containing one or more proteins of interest isadvantageous for a variety of reasons, including but not limited to, oneor more of the transient nature of the red blood cells and encodedproteins they will deliver; the lack of genetic material in the fullymature, anucleated cells; and the use of a “self” vessel that istolerated by the lymphoid compartment from ontogeny.

In some embodiments, proteins of interest are associated with red bloodcell membranes in vitro prior to administration, for example bytransfusion, into a subject. In some embodiments, such association ismediated by a covalent or non-covalent bond. Non-limiting examples ofsuch associations may include sulfhydryl bonds and antibody-epitopebinding, among others.

In some embodiments, proteins of interest are configured foradministration through incorporation into the outer surface membrane ofin vitro-produced red blood cells. In some embodiments, the HSC used inthe process of producing the red blood cell is engineered to expressand/or over-express one or more protein of interest. Optionally, theprotein of interest may be engineered with one or more of an exportsequence, a plasma membrane retention element, or a protease cleavagesite.

In some embodiments, proteins of interest are configured foradministration through incorporation into a red blood cell or HSC of thepresent disclosure using a fusion protein including a proteintransduction domain. Methods of making and using such PTD-protein ofinterest fusion proteins are known in the art and described herein, andinclude methods similar to those used for Myc and Bcl-2, among others.

In some embodiments, the HSCs are conditionally immortalized HSCs. Insome embodiments, the conditionally immortalized HSCs are proteintransduced HSCs, are conditionally immortalized through the use ofinducible transgenes, and/or are immortalized by the transientover-expression of one or more endogenous and/or exogenous genes thatpromote immortalization. Genes that promote immortalization are wellknown in the art (e.g., U.S. Patent Application Publication No. US2007/0116691).

In some embodiments, the conditionally immortalized HSCs are universaldonor HSCs. In some embodiments, the conditionally immortalized HSCs arefrom a rare blood type, or from an individual in need of treatment. Insome embodiments, the individual may suffer from a rare disease, have arare blood phenotype, or a blood phenotype for which it is difficult tofind an allotype match.

Construction of Vectors Encoding Proteins of Interest

In some aspects of the present disclosure, the proteins of interest areincorporated into HSCs of the present disclosure using any suitablemethod of transgenically modifying HSCs known in the art (e.g., Riviereet al., Blood. 2012 Feb. 2; 119(5):1107-16), including withoutlimitation, those disclosed herein. For example, one or more proteins ofinterest can be incorporated into HSCs utilizing methods similar tothose described herein to generate transgenic conditionally immortalizedHSCs (e.g., see “HSC cell line” section and Examples below).

In some embodiments, the proteins of interest will be encoded for by acDNA in the context of a vector, such as a viral vector. Methods forincorporating cDNA encoding proteins of interest into a viral vector areknown in the art and include, without limitation, those disclosedherein.

In some embodiments, a population of red blood cells of the presentdisclosure includes one or more proteins of interest designed to bereleased from the surface of the red blood cells following transfusionin vivo. In some embodiments, the vector will also include one or moreof an export sequence, a plasma membrane retention element, or aprotease cleavage site linked and/or fused with the protein of interest.

Protein export sequences are well known in the art. In some embodiments,the protein of interest will further include an endoplasmic reticulumsignal sequence, optionally at the N-terminal. In some embodiments, theprotein of interest is Bcl-2.

Plasma membrane retention elements are well known in the art, andinclude, without limitation, one or more of the cytoplasmic region of aGPI-linked protein, or optionally one or more red blood celltransmembrane protein such as CD40, CD25, igG, FcRN, CD8 and/or CD16.

Protease cleavage sites are well known in the art. In some embodiments,a plasma retention element may include transmembrane portions of IgGlinked to the protein of interest, where the IgG transmembrane and/orlinker portion includes one or more IgG cleavage sites, optionallyrecognized by mammalian proteases. The protease cleavage site may beselected for ease of release of the protein of interest in vivo,optionally through selection of a substrate of normal (endogenous) serumproteases.

In some embodiments, the population of red blood cells includes one ormore proteins of interest designed to be maintained on the surface ofthe red blood cell following transfusion in vivo. Accordingly, in someembodiments the vector includes one or more of an export sequence and aplasma membrane retention element. In some embodiments, the cytoplasmicand/or transmembrane portions of the fusion protein may be derived froma source such as, but not limited to, cDNA encoding one or more redblood cell transmembrane protein such as but not limited to CD40, CD25,igG, FcRN, CD8 and/or CD16, among others.

In some embodiments, the population of red blood cells includes one ormore proteins of interest designed to be maintained on the surface ofthe red blood cell and configured to bind proteins or other compoundsduring payloading prior to transfusion in vivo. This allows moreflexibility as far as payload options for the red blood cell, includingavoiding potential toxicity issues, and/or difficulties with geneticengineering of HSCs used for producing the red blood cells. In someembodiments, the vector will also include one or more of an exportsequence, a plasma membrane retention element, or a binding site orlinkage moiety. In some embodiments, binding sites or linkage moietiesmay include not are not limited to, sulfhydryl moieties, one portion ofa biotin-avidin linkage, as well as reversible and non-reversiblecrosslinking agents (e.g., DSS, DSST, PEG, etc.).

In some embodiments, it is explicitly contemplated to include one ormore proteins of interest in red blood cell or populations of red bloodcells of the present disclosure. For example, HSCs may be modified toincorporate targeting molecules, decoy receptors, and/or proteinpayloads, among others.

Exemplary Proteins of Interest

Although not intending to be limiting, a variety of examples of proteinsof interest and their use are provided. In some embodiments, the redblood cell-associated proteins of interest allow for the transientdelivery of hematopoietic growth factors and differentiation factorsincluding, but not limited to, EPO, TPO, and GM-CSF; or factors that maybe utilized to mobilize HSCs, such as G-CSF. In some embodiments,proteins of interest with targets in the hematopoietic compartment maybe incorporated into red blood cells using transgenic methods thatresult in the maintenance of the protein of interest on the surface ofthe red blood cell, thereby limiting uptake of the protein of interestinto other tissue compartments, and allowing more rapid clearance fromthe system.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of decoy receptors that affect inflammatorypathways in patients with acute inflammatory conditions, including, butnot limited to, IL-R-Fc, IL6R-Fc, TNFaR-FC, IFNaR-Fc, and BAFFR-Fc. Thered blood cells may be designed with one or more of these receptorsincorporated into their plasma membranes. Although not intending tobound by any theory, the receptors may be able to bind their cognateligands and remove the ligands from circulation, thereby decreasingthese circulating inflammatory mediators and ameliorating the acuteinflammatory condition. In some embodiments, a similar decoy receptorapproach may be used to remove other moieties from the blood, includingfor example, viral particles.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of decoy ligands that can competitively bind toreceptors associated with cytokine storms, including, but not limitedto, IL-R-Fc, IL6R-Fc, TNFaR-FC, IFNaR-Fc, and BAFFR-Fc, and alleviatethe clinical signs associated with a cytokine storm using a similarapproach.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of protein-encoded toxins, including, but notlimited to, cholera toxin, shigella toxin, ricin, and diphtheria toxin.Such toxic payloads may be designed for the treatment of cancers, suchas solid tumors that are highly vascularized. In some embodiments, theproteins of interest can be engineered to be expressed on these redblood cells. In some embodiments, HSCs are engineered to expresstransmembrane proteins having an external portion configured with acovalent or non-covalent binding site. For example, the external portionmay have sulfhydryl moieties designed to form disulfide bonds,avidin-biotin linkages, or any other linkage that allows the laterassociation of the desired payload (e.g., protein-encoded toxins, andoptionally other desired payloads). For example, the red blood cells mayalso be engineered using one or more of these approaches to delivermembrane-bound angiogenic inhibitors to the sites of the tumors.

In some embodiments, the red blood cell-associated proteins of interestallow for the targeting of the red blood cells to tissues and/or cellsof interest. For example, targeting may be useful in bringing red bloodcells to tumors or to tumor cells, among other uses. In someembodiments, targeting may be achieved through a variety of methodsincluding, but not limited to, expression of markers, receptors,ligands, and antibodies on the surface of the red blood cells, orthrough linkage of one or more of these moieties onto the surface of thered blood cells.

In some embodiments, the red blood cell associated proteins of interestallow for the delivery of proangiogenic and/or lymphangiogenic factorsto tissues affected by vascular dysregulation such as but not limited tofrost bite, cancer-related vasoconstriction, or rheumatic joints, etc.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of vasodilatory peptides in acute cases involvingvassal constriction, including, but not limited to, vessel constrictionduring acute cardiac infarctions, obstetrical uses during childdelivery, and acute and/or persistent migraine headaches, among others.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of antigens for boosting life-long immunity. Theantigen may be delivered via a genetically modified red blood celldesigned to present the antigen of interest (protein) along with a TLRligand on the red blood cell.

In some embodiments, the red blood cell-associated proteins of interestallow for the delivery of one or more proteins of interest to alleviateclot formation in patients at high risk of having clots. In someembodiments, a blood transfusion to a patient at high risk of havingclots may include red blood cells having membrane-bound proteases thatare specific to fibrotic tissues in embolitic masses, and optionallyhaving targeting molecules to such locations. A similar approach may beuseful for patients at elevated risk for pulmonary embolisms.

In some embodiments, the proteins of interest do not include Myc orBcl-2.

Protein Transduction Domains

As used herein, the terms “peptide transduction domain,” “proteintransduction domain,” and “PTD” are used interchangeably and refer to apeptide sequence or domain of a protein that promotes penetration ofprotein into a mammalian cell and/or compartment(s) within a mammaliancell. In one non-limiting example, a PTD promotes penetration of acoupled peptide and/or protein into the nucleus of a cell.

PTDs of the present disclosure may be isolated from a PTD-containingprotein by any method of isolating a protein domain known in the art,such as standard molecular biology and biochemical techniques.Alternatively, PTDs of the present disclosure may be synthesized.Suitable PTDs of the present disclosure may be about 8 to about 30 aminoacid residues in length, and enriched in basic amino acid residues, suchas argentine (Arg) and lysine (Lys). In some embodiments, PTDs may havea short peptide sequence enriched in basic amino acids (arginine andlysine), optionally arranged in an alpha-helical structure.

As disclosed herein, PTDs of the present disclosure are coupled (e.g.,fused, conjugated, cross-linked, etc.) to a peptide and/or protein inorder to facilitate the penetration of the peptide and/or protein into amammalian cell and/or compartment within a mammalian cell. For example,in certain embodiments a PTD of the present disclosure is coupled to aMYC protein and/or a Bcl-2 protein and/or a protein of interest.

Protein transduction domains suitable for use in any of the methods ofthe present disclosure include any PTD known in the art (e.g., U.S.Patent Application Publication Nos. US 2007/0116691 and US2010/0055129). For example, suitable PTDs may be obtained or derivedfrom proteins that include, without limitation, lentiviral TAT(Trans-Activator of Transcription) proteins, lentiviral VPR proteins,herpesviral VP22 proteins, and homeoproteins.

Examples of suitable PTDs obtained or derived from lentiviral TATproteins include, without limitation, the PTD from a TAT protein of aTAT protein-containing virus, the PTD from a TAT protein of a TATprotein-containing lentivirus, the PTD from the HIV-1 TAT protein, thePTD from the HIV-2 TAT protein, the PTD from the SIV TAT protein, thePTD from a primate lentivirus TAT protein, the PTD from an ovinelentivirus TAT protein, the PTD from a bovine lentivirus TAT protein,the PTD from an equine lentivirus TAT protein, the PTD from a felinelentivirus TAT protein, a PTD from the TAT protein of a subvariant ofHIV, SIV, primate lentivirus, ovine lentivirus, bovine lentivirus,equine lentivirus, or feline lentivirus, and homologues thereof. Incertain embodiments, the PTD is amino acid residues 48-57 of the HIV TATprotein (TAT_([48-57])). In other embodiments, the PTD is amino acidresidues 57-48 of the HIV TAT protein (TAT_([57-48])).

Examples of suitable PTDs that may obtained or derived from lentiviralVPR proteins include, without limitation, the PTD from a VPR protein ofa VPR protein-containing virus, the PTD from a VPR protein of a VPRprotein-containing lentivirus, the PTD from the HIV-1 VPR protein, thePTD from the HIV-2 VPR protein, the PTD from the SIV VPR protein, thePTD from a primate lentivirus VPR protein, the PTD from an ovinelentivirus VPR protein, the PTD from a bovine lentivirus VPR protein,the PTD from an equine lentivirus VPR protein, the PTD from a felinelentivirus VPR protein, a PTD from the VPR protein of a subvariant ofHIV, SIV, primate lentivirus, ovine lentivirus, bovine lentivirus,equine lentivirus, or feline lentivirus, and homologues thereof.

Examples of suitable PTDs that may obtained or derived from herpesviralVP22 proteins include, without limitation, the PTD from the humanherpesvirus 1 (HSV-1) VP22 protein, the PTD from the human herpesvirus 2(HSV-2) VP22 protein, the PTD from the BHV-1 VP22 protein, the PTD fromthe Psittacid herpesvirus 1VP22 protein, the PTD from the Equineherpesvirus 1 VP22 protein, the PTD from the Equine herpesvirus 4 VP22protein, the PTD from the Gallid herpesvirus 2 VP22 protein, the PTDfrom the Varicella-zoster virus VP22 protein, and homologues thereof.

Examples of suitable PTDs that may be obtained or derived fromhomeodomain transcription factors include, without limitation, thehomeodomain (HD) from the Drosophila Antennapedia (Antp) protein, the HDfrom the Drosophila Fushi tarazu (Ftz) protein, the HD from theDrosophila Engrailed (En) protein, the HD from the chick Engrailed-2protein, the HD from mammalian homeoproteins, the HD from humanhomeoproteins, the HD from human Hox-A5 homeoprotein, the HD from humanHox-A4 homeoprotein, the HD from human Hox-B5 homeoprotein, the HD fromhuman Hox-B6 homeoprotein, the HD from human Hox-B7 homeoprotein, the HDfrom human HOX-D3 homeoprotein, the HD from human GOX homeoprotein, theHD from human MOX-2 homeoprotein, the HD from human Hoxc-8 homeoprotein,the HD from human Islet-1 (Isl-1) homeoprotein, and homologues thereof.

Additionally, suitable PTDs include, without limitation, the PTD derivedfrom Kaposi-FGF (K-FGF or FGF-4), the PTD derived from FGF-2, the PTDderived from FGF-1, and the PTD from other members of the FGF-family ofproteins.

Other suitable PTDs include synthetic PTDs (e.g., Beerens, A M J et al.Curr Gene Ther. 2003 October; 3(5):486-94). In some embodiments, asynthetic PTD may include EPTD, an optimized protein transduction domain(YARAAARQARA) (Ho, A. et al., Cancer Res. (2001) 61:474-477).

Further suitable PTDs include, without limitation, a CHARIOT™ peptide(Active Motif, Carlsbad, Calif.).

In some embodiments, PTDs of the present disclosure are producedrecombinantly, while in others the PTDs are produced synthetically orare purified from a native source.

PTD Fusion Protein Modifications

In some embodiments, PTD fusion proteins of the present disclosurecontain one or more molecules that link the PTD to a polypeptide, suchas a recombinant protein of the present disclosure that promotes cellsurvival and/or proliferation, an exogenous protein of the presentdisclosure that inhibits apoptosis, or a protein of interest of thepresent disclosure. In further embodiments, the one or more linkermolecules are amino acid peptides.

PTD fusion proteins of the present disclosure may further contain atleast one amino acid sequence that facilitates purification of thefusion proteins. For example, the PTD fusion proteins may contain aprotein tag, such as a polyhistidine tag. Alternatively, or in addition,the PTD-MYC fusion proteins may contain an epitope tag, such as a V5epitope tag.

Accordingly, in certain embodiments, PTD fusion proteins of the presentdisclosure further contain a polyhistidine tag. In some embodiments, thepolyhistidine tag is a 6-histidine tag. In some embodiments, thehistidine tag contains the sequence HHHHHH. Additionally, the histidinetag may be added to a PTD fusion protein of the present disclosure byany suitable method known in the art. For example, a PTD fusion proteinsequence may be cloned into an expression vector encoding apolyhistidine tag. Alternatively, a polyhistidine tag may be added byPCR (i.e., the PCR primers contain a polyhistidine sequence).

Moreover, a PTD fusion protein of the present disclosure may alsocontain at least one protein tag. In some embodiments, the at least oneprotein tag is an epitope tag. Preferably, the epitope tag is a V5epitope tag. In some embodiments, the V5 epitope tag contains the aminoacid sequence: GKPIPNPLLGLDST, while in other embodiments the V5 epitopetag contains the amino acid sequence: IPNPLLGLD. The amino acids may beeither in the D formation, or in the L formation. In some embodiments, afirst plurality of amino acids is in the D formation and a secondplurality is in the L formation. Additionally, aV5 epitope tag of thepresent disclosure may be added to a PTD fusion protein of the presentdisclosure by any suitable method known in the art. For example, a PTDfusion protein sequence may be cloned into an expression vector encodinga V5 epitope tag. Alternatively, aV5 epitope tag may be added by PCR(i.e., the PCR primers contain a V5 epitope sequence).

In certain preferred embodiments, a PTD fusion protein of the presentdisclosure further contains a polyhistidine tag and an epitope tag.Preferably, the PTD fusion protein contains a 6-histidine tag and a V5epitope tag.

Construction of PTD Fusion Proteins

In some embodiments, a PTD fusion protein of the present disclosure maybe constructed by any suitable method known in the art (e.g., U.S.Patent Application Publication No. US 2010/0055129).

In one non-limiting example, a nucleic acid sequence encoding aPTD-recombinant protein that promotes cell survival and/or proliferationof the present disclosure (e.g., a PTD-MYC fusion protein, a PTD-ICN-1fusion protein, etc.) may be generated by PCR. In certain embodiments,nucleic acid sequence encoding a PTD-MYC fusion protein is generated byPCR. This may be accomplished by designing a forward primer for a MYCsequence that contains an in frame PTD sequence, such as the RKKRRQRRR9-amino-acid sequence of TAT, and a reverse primer for the MYC sequencethat is designed to remove the stop codon. The PCR product from a PCRreaction using such primers may then be cloned into any suitableexpression vector known in the art.

In one non-limiting example, a nucleic acid sequence encoding aPTD-exogenous protein that inhibits apoptosis of the present disclosure(e.g., a PTD-Bcl-2 fusion protein, a PTD-Bcl-w fusion protein, aPTD-Bcl-X fusion protein, a PTD-Bcl-X1 fusion protein, a PTD-Mcl-1fusion protein, etc) may be generated by PCR. In certain embodiments,nucleic acid sequence encoding a PTD-Bcl-2 fusion protein is generatedby PCR. This may be accomplished by designing a forward primer for aBcl-2 sequence that contains an in frame PTD sequence, such as theRKKRRQRRR 9-amino-acid sequence of TAT, and a reverse primer for theBcl-2 sequence that is designed to remove the stop codon. The PCRproduct from a PCR reaction using such primers may then be cloned intoany suitable expression vector known in the art. The Bcl-2 unstructuredloop may be removed from the BCL-2 coding sequence using a site directedmutagenesis kit.

In one non-limiting example, a nucleic acid sequence encoding aPTD-protein of interest fusion protein of the present disclosure may begenerated by PCR. This may be accomplished by designing a forward primerfor a protein of interest sequence that contains an in frame PTDsequence, such as the RKKRRQRRR 9-amino-acid sequence of TAT, and areverse primer for the protein of interest sequence that is designed toremove the stop codon. The PCR product from a PCR reaction using suchprimers may then be cloned into any suitable expression vector known inthe art.

Hematopoietic Stem Cells

Other aspects of the present disclosure relate to the in vitroproduction of a population of red blood cells by culturing hematopoieticstem cells (HSCs), optionally conditionally immortalized and/orgenetically engineered to include one or more protein of interest, withEPO, and optionally IL-3, and one or more recombinant protein, orbiologically active fragment thereof, that promotes cell survival and/orproliferation. This process may be performed in the presence or theabsence of feeder cells and serum. In certain preferred embodiments, theprocess is performed in the absence of feeder cells and/or serum.

HSCs suitable for use with the methods of the present disclosure may beproduced from embryonic stem (ES) cells and/or induced pluripotent stem(iPS) cells. Any method of producing HSCs from ES cells and/or iPS cellsknown in the art may be used (e.g., Keller, G. Genes Dev. 2005 19:1129-1155; and Papapetrou Sadelain, F1000 Med Rep. 2010 Jun. 16; 2). Forexample, HSCs may be produced from ES cells by patterning thehematopoietic development of ES cell culture on the hematopoieticcommitment in the early embryo (e.g., Keller, G. Genes Dev. 2005 19:1129-1155).

Additionally, HSCs suitable for use with the methods of the presentdisclosure may be obtained by any suitable technique known in the art.For example, HSCs may be found in the bone marrow of a donor, whichincludes femurs, hip, ribs, sternum, and other bones. Any method knownin the art for extracting or harvesting bone marrow cells may be used.In one non-limiting example, HSCs may be obtained directly from themarrow cavity of the hip using a needle and syringe to aspirate cellsfrom the marrow cavity. Rich marrow may be obtained from the hip byperforming multiple small aspirations.

Alternatively, suitable HSCs may be obtained from peripheral blood cellsfound in the blood of a donor, optionally following pre-treatment withcytokines, such as G-CSF (granulocyte colony-stimulating factors), thatinduce HSCs to be released from the bone marrow compartment of thedonor. HSCs may also be obtained from peripheral blood that hasundergone an apheresis procedure to enrich for HSCs. Any apheresisprocedure known in the art may be used. In certain embodiments, theapheresis procedure is a leukapheresis procedure.

Additionally, suitable HSCs may be obtained from umbilical cord blood,placenta, and mobilized peripheral blood. For experimental purposes,fetal liver, fetal spleen, and AGM (Aorta-gonad-mesonephros) of animalsare also useful sources of HSCs. Additionally, HSCs may be procured froma source that obtained HSCs from the bone marrow, peripheral blood,umbilical cord, or fetal tissue of a donor.

In some embodiments, HSCs are obtained from a human umbilical cord orplacenta. Another source of HSCs that may be utilized is the developingblood-producing tissues of fetal animals. In humans, HSCs may be foundin the circulating blood of a human fetus by about 12 to 18 weeks. Insome embodiments, human HSCs are obtained from any source, e.g., thebone marrow, umbilical cord, peripheral blood, or fetal tissue of blood,of type A+, A−, B+, B−, O+, O−, AB+, and AB− donors. In otherembodiments, human HSCs are obtained from any source, e.g., the bonemarrow, umbilical cord, peripheral blood, or fetal tissue of blood, ofuniversal donors or donors having a rare blood type. Rare blood typesare know in the art and include, without limitation, Oh, CDE/CDE,CdE/CdE, C^(W)D−/C^(W)D−, −D−/−D−, Rh_(null), Rh: −51, LW(a−b+),LW(a−b−), S−s−U−, S−s−U(+), pp, Pk, Lu(a+b−), Lu(a−b−), Kp(a+b−),Kp(a−b−), Js(a+b−), Ko, K: −11, Fy(a−b−), Jk(a−b−), Di(b−), I−, Yt(a−),Sc: −1, Co(a−), Co(a−b−), Do(a−), Vel−, Ge−, Lan−, Lan(+), Gy(a−), Hy−,At(a−), Jr(a−), In(b−), Tc(a−), Cr(a−), Er(a−), Ok(a−), JMH−, andEn(a−).

In other embodiments, human HSCs are obtained from any source, e.g., thebone marrow, umbilical cord, peripheral blood, or fetal tissue of blood,of donors having an auto-immune disorder, immune deficiency, or anyother disease or disorder that would benefit from a transplantation ofHSCs and/or transfusion of blood. Such donors may also be therecipients. Advantageously, HSCs obtained from such donor may be usedfor personalized HSC and/or blood therapy.

In one non-limiting example, human HSCs may be obtained by anesthetizingthe stem cell donor, puncturing the posterior superior iliac crest witha needle, and performing aspiration of bone marrow cells with a syringe.In another non-limiting example, HSCs may be obtained from theperipheral blood of a donor, where a few days prior to harvesting thestem cells form the peripheral blood, the donor is injected with G-CSFin order to mobilize the stem cells to the peripheral blood.

Accordingly, in some embodiments, HSCs are obtained from an autologousdonor, that is the donor will also be the recipient of the HSCs and/orred blood cells derived from such HSCs. Any methods known in the art anddescribed herein may be used to obtain HSCs from the autologous donor.The HSCs and/or any therapeutic products derived or produced therefrom,such as red blood cells, are then transplanted, administered, and ortransfused back to the original donor. Similarly, HSCs may be obtainedfrom an allogenic donor, such as a sibling, parent, or other relative ofa subject in need of an HSC transplantation and/or blood transfusion. Inone non-limiting example, allogenic HSCs are obtained by collecting HSCsfrom different blood groups or major histocompatibility complex (MHC) orhuman leukocyte antigen (HLA) matching sources. Autologous and/orallogenic HSC transplantation and/or blood transfusion may occur at anytime after the donation, such as days later, months later, or even yearslater. Autologous donation may be particularly useful in cases where thesubject in need of HSCs and/or blood transplantation and/or transfusionwould have a negative, deleterious, or toxic reaction to transplantationand/or transfusion of HSCs and/or blood from any other donor, includingallogenic and/or universal donors. Examples of patients that may benefitfrom autologous and/or allogenic donation are well known in the art andinclude, without limitation, those suffering from an autoimmunedisorder, blood disease or disorder, immune disease or disorder, orother related diseases or conditions.

Cells obtained from, for example, bone marrow, peripheral blood, or cordblood, are typically processed after extraction or harvest. Any methodknown in the art for processing extracted or harvested cells may beused. Examples of processing steps include, without limitation,filtration, centrifugation, screening for hematopathologies, screeningfor viral and/or microbial infection, erythrocyte depletion, T-celldepletion to reduce incidence of graft-versus-host disease in allogenicstem cell transplant recipients, volume reduction, cell separation,resuspension of cells in culture medium or a buffer suitable forsubsequent processing, separation of stem cells from non-stem cells(e.g., stem cell enrichment), ex vivo or in vitro stem cell expansionwith growth factors, cytokines, and/or hormones, and cryopreservation.

Any suitable method for stem cell enrichment known in the art may beused. Examples of stem cell enrichment methods include, withoutlimitation, fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS).

Accordingly, in certain embodiments, HSCs suitable for use in themethods of the present disclosure are human HSCs.

HSCs obtained from a donor may be identified and/or enriched by anysuitable method of stem cell identification and enrichment known in theart, such as by utilizing certain phenotypic or genotypic markers. Forexample, in some embodiments, identification of HSCs includes using cellsurface markers associated with HSCs or specifically associated withterminally differentiated cells of the system. Suitable surface markersmay include, without limitation, one or more of c-kit, Sca-1, CD4, CD34,CD38, Thy1, CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105,CD133, CD135, ABCG2, NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin,Gr-1, CD46, Mac-1, Thy1.1, and the signaling lymphocyte activationmolecule (SLAM) family of receptors. Examples of SLAM receptors include,without limitation, CD150, CD48, and CD244.

Additionally, HSCs obtained from a donor may be separated from non-stemcells by any suitable method known in the art including, withoutlimitation, fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS).

In one non-limiting example, human peripheral blood cells are incubatedwith antibodies recognizing c-kit, Sca-1, CD34, CD38, Thy1, CD2, CD3,CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105, CD133, ABCG2, NK1.1, B220,Ter-119, Flk-2, CDCP1, Endomucin, or Gr-1. Antibodies for CD2, CD3, CD4,CD5, CD8, NK1.1, B220, Ter-119, and Gr-1 are conjugated with magneticbeads. The cells expressing CD2, CD3, CD4, CD5, CD8, NK1.1, B220,Ter-119, or Gr-1 are retained in the column equipped to trap magneticbeads and cells attached to magnetic bead conjugated antibodies. Thecells that are not captured by the MACS column are subjected to FACSanalysis. Antibodies for c-kit, Sca-1, CD34, CD38, Thy1, are conjugatedwith fluorescent materials known in the art. The cells that are CD34⁺,CD38^(low/−), c-kit^(−/low), Thy1⁺ are separated from the rest of sampleby virtue of the types of fluorescent antibodies associated with thecells. These cells are provided as human long-term HSCs suitable for usewith any of the methods of the present disclosure.

In another non-limiting example, cells obtained from a subject arelabeled with the same set of magnetic bead conjugated antibodies asdescribed above (antibodies against one or more of CD2, CD3, CD4, CD5,CD8, NK1.1, B220, Ter-119, or Gr-1) and fluorescent conjugated CD 150,CD244 and/or CD48 antibodies. After removing cells captured by themagnetic bead conjugated antibodies from the sample, the sample isanalyzed by FACS and CD150⁺, CD244⁻ and CD48⁻ cells are retained aslong-term HSCs.

In some embodiments, HSCs utilized in the methods of the presentdisclosure contain one or more of the markers: c-kit⁺, Sca-1⁺,CD34^(low/−), CD38⁺, Thy1^(+/low), CD34⁺, CD38^(low/−), c-kit^(−/low),and/or Thy1⁺. In some embodiments, the HSCs utilized in the methods ofthe present disclosure lack one or more of the markers: CD2, CD3, CD4,CD5, CD8, NK1.1, B220, Ter-119, and/or Gr-1. In certain embodiments, theHSCs utilized in the methods of the present disclosure are of an A+, A−,B+, B−, O+, O−, AB+, or AB− type.

Alternatively, suitable HSCs may be obtained from a non-human source.Suitable non-human HSCs may be isolated from, femurs, hip, ribs,sternum, and other bones of a non-human animal, including, withoutlimitation, laboratory/research animals, rodents, pets, livestock, farmanimals, work animals, pack animals, rare or endangered species, racinganimals, and zoo animals. Further examples of suitable non-human animalsinclude, without limitation, monkies, primates, mice, rats, guinea pigs,hamsters, dogs, cats, horses, cows, pigs, sheep, goats, and chickens.For example, HSCs may be obtained from murine bone marrow cells, byincubating the bone marrow cells with antibodies recognizing cellsurface molecules such as one or more of c-kit, Sca-1, CD34, CD38, Thy1,CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105, CD133, ABCG2,NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin, or Gr-1. Antibodies forCD2, CD3, CD4, CD5, CDS, NK1.I, B220, Ter-119, and Gr-1 are conjugatedwith magnetic beads. In MACS equipment, the cells harboring CD2, CD3,CD4, CD5, CD8, NK1.1, B220, Ter-119, or Gr-1 on their surface areretained in the column equipped to trap magnetic beads and the cellsattached to magnetic bead conjugated antibodies. The cells that are notcaptured by MACS column are subjected to FACS analysis. For FACSanalysis, Antibodies for surface molecules such as c-kit, Sca-1, CD34,CD38, Thy1, are conjugated with fluorescent materials. The cells thatare c-kit⁺, Sca-1⁺, CD34^(low/−), CD38⁺, Thy1^(+/low) are separated fromthe rest of the sample by virtue of the types of fluorescent antibodiesassociated with the cells. These cells are provided as murine long-termHSCs suitable for use with any of the methods of the present disclosure.In other embodiments, different sets of marker are used to separatemurine long-term HSCs from cells of bone marrow, umbilical cord blood,fetal tissue, and peripheral blood.

In some embodiments, obtaining HSCs from bone marrow includes firstinjecting the HSC donor, such as a mouse or other non-human animal, with5-fluorouracil (5-FU) to induce the HSCs to proliferate in order toenrich for HSCs in the bone marrow of the donor.

Moreover, HSCs suitable for use with any of the methods of the presentdisclosure, whether obtained from, or present in, cord blood, bonemarrow, peripheral blood, or other source, may be grown or expanded inany suitable, commercially available or custom defined medium (e.g.,Hartshorn et al., Cell Technology for Cell Products, pages 221-224, R.Smith, Editor; Springer Netherlands, 2007). For example, serum freemedium may utilize albumin and/or transferrin, which have been shown tobe useful for the growth and expansion of CD34⁺ cells in serum freemedium. Also, cytokines may be included, such as Flt-3 ligand, stem cellfactor (SCF), and thrombopoietin (TPO), among others. HSCs may also begrown in vessels such as bioreactors (e.g., Liu et al., Journal ofBiotechnology 124:592-601, 2006). A suitable medium for ex vivoexpansion of HSCs may also contain HSC supporting cells, such as stromalcells (e.g., lymphoreticular stromal cells), which can be derived, forexample, from the disaggregation of lymphoid tissue, and which have beenshown to support the in vitro, ex vivo, and in vivo maintenance, growth,and differentiation of HSCs, as well as their progeny.

HSC growth or expansion may be measured in vitro or in vivo according toroutine techniques known in the art. For example, WO 2008/073748,describes methods for measuring in vivo and in vitro expansion of HSCs,and for distinguishing between the growth/expansion of HSCs and thegrowth/expansion of other cells in a potentially heterogeneouspopulation (e.g., bone marrow), including for example intermediateprogenitor cells.

HSC Cell Lines

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure may also be derived from an HSC cell line. SuitableHSC cell lines include any cultured hematopoietic stem cell line knownin the art. Non-limiting examples include the conditionally immortalizedlong-term stem cell lines described in U.S. Patent ApplicationPublication Nos. US 2007/0116691 and US 2010/0047217.

In certain embodiments, HSCs suitable for use in the methods of thepresent disclosure are conditionally immortalized before beingdifferentiated into red blood cells. In some embodiments, HSCs suitablefor use in the methods of the present disclosure may also be modified toinclude one or more proteins of interest before being differentiatedinto red blood cells. In some embodiments, conditional immortalizationand/or inclusion of one or more proteins of interest may be achievedthrough any method known in the art and described herein, such as one ormore of a transgenic approach, a protein-transduction approach, or anapproach enhancing the expression of endogenous proteins.

Proteins Useful for Conditionally Immortalizing HSCs

In some embodiments, HSCs used in the methods of the present disclosurefor the production of red blood cells were conditionally immortalized bycontacting the HSCs with a composition containing one or morerecombinant protein that promotes cell survival and/or cellproliferation and, optionally, one or more exogenous protein thatinhibits apoptosis. In some embodiments, the one or more recombinantprotein that promotes cell survival and/or proliferation also optionallyinhibits apoptosis. In some embodiments, the one or more recombinantprotein that promotes cell survival and/or proliferation is a MYCpolypeptide, a biologically active fragment thereof or homologuethereof, of the present disclosure. In some embodiments, the one or morerecombinant protein that promotes cell survival and/or proliferationfurther includes a protein transduction domain (PTD). In someembodiments, the one or more recombinant protein that promotes cellsurvival and/or proliferation is a PTD-MYC fusion protein. In someembodiments, the one or more exogenous protein that inhibits apoptosisis a protein that includes a Bcl-2 homology domain. In some embodiments,the one or more of exogenous protein that inhibits apoptosis furtherinclude a protein transduction domain (PTD). In some embodiments, theone or more of exogenous protein that inhibits apoptosis is a PTD-Bcl-2fusion protein.

PTD-MYC and PTD-Bcl-2 fusion proteins of the present disclosure allowfor an increase in MYC and Bcl-2 activity in HSCs by the exogenousaddition of MYC and Bcl-2, without the need for overexpressing theendogenous genes encoding MYC and Bcl-2, or recombinantly expressing MYCand Bcl-2 via genetic manipulation. However, manipulation of HSCs toinduce overexpression of such endogenous genes, optionally, through thecreation of transgenic HSCs as well as other techniques to createconditionally immortalized stem cells are expressly envisioned herein.

In some embodiments, the HSCs to be differentiated into red blood cells(and/or the red blood cells) are further transduced with one or moredisclosed proteins of interest. In some embodiments, these one or moreproteins of interest are other than Myc or Bcl-2. In some embodiments,the proteins of interest are fusion proteins including a PTD domain ofthe present disclosure. In some embodiments, the PTD domain is TAT.

Transgenic Approach

In some embodiments, conditionally immortalized HSCs for use in themethods of the present invention are established using any transgenicapproach known in the art (e.g., U.S. Patent Application PublicationNos. US 2007/0116691 and US 2010/0047217. For example, HSCs may beimmortalized by obtaining an expanded population of HSCs, transfecting(transducing) the HSCs with a vector that encodes a recombinant proteinthat promotes cell survival and/or proliferation that is regulatable(e.g., inducible and/or controllable), transfecting (transducing) theHSCs with a vector encoding a recombinant protein that inhibitsapoptosis, and expanding the transfected HSCs in the presence of acombination of stem cell growth factors under conditions where therecombinant protein that promotes cell survival and/or proliferation isinduced and/or active.

The recombinant protein that promotes cell survival and/or proliferationis regulatable (e.g., inducible or controllable), so that theprecombinant protein can be activated and deactivated (i.e., turned onor turned off) as desired to either maintain the HSCs in an immortalizedstate or to allow it to differentiate into a desired cell type, such asa red blood cell. The recombinant protein that promotes cell survivaland/or proliferation may be any protein of the present disclosure thatpromotes cell survival and/or proliferation. In certain preferredembodiments, the protein that promotes cell survival and/orproliferation is MYC. Similarly, the recombinant protein that inhibitsapoptosis may be any protein of the present disclosure that inhibitsapoptosis. In certain preferred embodiments, the protein that inhibitsapoptosis is Bcl-2.

In some embodiments, the recombinant protein that promotes cell survivaland/or proliferation and/or the recombinant protein that inhibitsapoptosis has been modified such that activity is inducible orrepressible. For example, the recombinant proteins may further containan inducible receptor. In certain embodiments, the recombinant proteinscontain an estrogen receptor (ER). In certain embodiments, therecombinant protein that promotes cell survival and/or proliferation andthat contains an estrogen receptor is a MYC-ER polypeptide. In certainembodiments, the recombinant protein that inhibits apoptosis and thatcontains an estrogen receptor is a Bcl-2-ER polypeptide. In certainembodiments, the recombinant proteins containing an estrogen receptorare induced by 4-hydroxytamoxifen (4-OHT). Alternatively, therecombinant proteins may contain a glucocorticoid receptor (GR), e.g., aglucocorticoid receptor that is sensitive to mifepristone (MIFEPREX). Incertain embodiments, the recombinant protein that promotes cell survivaland/or proliferation and that contains a glucocorticoid receptor is aMYC-GR polypeptide. In certain embodiments, the recombinant protein thatinhibits apoptosis and that contains a glucocorticoid receptor is aBcl-2-GR polypeptide.

Any method known in the art for obtaining an expanded population of HSCsknown in the art may be used. For example, HSCs may be cultured with oneor more growth factor that promotes cell proliferation and/or celldivision.

Preferably, the vectors are an integrating vector, which has the abilityto integrate into the genome of a cell (e.g., a retroviral vector). TheHSCs can be transfected and/or transduce with the vectors using anysuitable method of transfecting cells, and particularly mammalian cells,including by using combinations of techniques. Examples of suitablevectors, include without limitation, retroviral vectors, lentivirusvectors, parvovirus vectors, vaccinia virus vectors, coronavirusvectors, calicivirus vectors, papilloma virus vectors, flavivirusvectors, orthomixovirus vectors, togavirus vectors, picornavirusvectors, adenoviral vectors, and modified and attenuated herpesvirusesvectors. Any such virus vector can further be modified with specificsurface expressed molecules that target these to HSCs, such as membranebound SCF, or other stem-cell specific growth factor ligands. Othermethods of transfection of mammalian cells include, but are not limitedto, direct electroporation of mammalian expression vectors, such as byusing NUCLEOFECTOR™ technology (AMAXA Biosystems). This technology is ahighly efficient non-viral gene transfer method for most primary cellsand for hard-to-transfect cell lines, which is an improvement on thelong-known method of electroporation, based on the use of cell-typespecific combinations of electrical current and solutions to transferpolyanionic macromolecules directly into the nucleus. Additionally,suitable methods of transfection can include any bacterial, yeast, orother artificial methods of gene delivery that are known in the art.

In some embodiments, one or more of the proteins of interest areincorporated into one or more HSCs suitable for use in the methods ofthe present disclosure (e.g., primary cell lines or conditionallyimmortalized cell lines) using approaches similar to those described forthe production of conditionally immortalized HSCs.

Enhancement of Endogenous Expression

In some embodiments, conditionally immortalized HSCs for use in themethods of the present disclosure may be established by enhancing theexpression of endogenous proteins that promote cell survival and/orproliferation, including, without limitation, any protein of the presentdisclosure that promote cell survival and/or proliferation. For example,expression of an endogenous onco-peptide of the present disclosure, MYCpolypeptide, ICN-1 polypeptide, homologue thereof, and/or analoguethereof may be enhanced. Additionally, conditionally immortalized HSCsfor use in the methods of the present disclosure may be established byalso enhancing the expression of endogenous proteins that inhibitapoptosis, including, without limitation, any protein of the presentdisclosure that inhibits apoptosis. For example, expression of anendogenous protein of the present disclosure that contains one or moreBcl-2 homology domain of the present disclosure, Bcl-2 polypeptide,Bcl-x polypeptide, Bcl-XL polypeptide, Mcl-1 polypeptide, CED-9polypeptide, Bcl-2 related protein A1 polypeptide, Bfl-1 polypeptide,Bcl-w polypeptide, homologue thereof, and/or analogue thereof may beenhanced.

In some embodiments, the expression of one or more of the proteins ofinterest is increased in one or more HSCs suitable for use in themethods of the present disclosure (e.g., primary cell lines orconditionally immortalized cell lines) using approaches similar to thosedescribed for the enhanced production of, for example, MYC and/or Bcl-2.

Protein Transduction Approach

In some embodiments, HSCs obtained and/or produced by any methoddisclosed herein may be treated with a gene product that promotes cellsurvival and/or proliferation, including, but not limited to anyrecombinant protein of the present disclosure that promotes cellsurvival and/or proliferation (e.g., onco-peptide, MYC, ICN-1,homologues thereof, analogues thereof, and biologically active fragmentsthereof) and/or with a protein of the present disclosure that inhibitsapoptosis of the HSCs (e.g., proteins containing one or more Bcl-2homology domains, Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 relatedprotein A1, Bfl-1, Bcl-w, homologues thereof, analogues thereof, andbiologically active fragments thereof) (e.g., U.S. Patent ApplicationPublication No. US 2007/0116691). In some embodiments, the protein thatpromotes cell survival and/or proliferation is a fusion proteincontaining a PTD. In some embodiments, the protein that inhibitsapoptosis is a fusion protein containing a PTD. In some embodiments,HSCs obtained and/or produced by any method disclosed herein may betreated with one or more compound (optionally an exogenous protein) thatenables the transient upregulation of at least one function of arecombinant protein of the present disclosure that promotes cellsurvival and/or proliferation in a cell. In some embodiments, therecombinant protein that promotes cell survival and/or proliferationprotein is a PTD-MYC and/or ICN-1 fusion protein. In certainembodiments, the PTD-MYC fusion protein is a TAT-MYC and/or ICN-1 fusionprotein.

In some embodiments, HSCs obtained by any method disclosed herein may betreated with one or more compound (optionally an exogenous protein) thatenables the transient upregulation of at least one function of arecombinant protein of the present disclosure that inhibits apoptosis ina cell. In some embodiments, the exogenous protein that inhibitsapoptosis of the HSCs is a PTD-Bcl-2 fusion protein. In someembodiments, the PTD-Bcl-2 fusion protein is a TAT-Bcl-2 fusion protein.

In some embodiments, one or more of the proteins of interest areincorporated into one or more HSCs suitable for use in the methods ofthe present disclosure (e.g., primary cell lines or conditionallyimmortalized cell lines) using approaches similar to those described forthe production of conditionally immortalized HSCs using PTD-fusionproteins.

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure are contacted with a composition containing a fusionprotein containing a protein of the present disclosure that promotescell survival and/or proliferation fused to a PTD (e.g., a PTD-MYCand/or PTD-ICN-1 fusion protein). In further embodiments, thecomposition further contains a fusion protein containing a protein ofthe present disclosure that inhibits apoptosis fused to a PTD (e.g., aPTD-Bcl-2 fusion protein). In some embodiments, the HSCs are contactedwith a composition containing a PTD-MYC and/or PTD-ICN-1 fusion protein,and a second composition containing a PTD-Bcl-2 fusion protein.

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure are expanded in the presence of a fusion proteincontaining a protein of the present disclosure that promotes cellsurvival and/or proliferation fused to a PTD (e.g., a TAT-MYC proteinand/or TAT-ICN-1 protein fusion) prior to being differentiated into redblood cells. In further embodiments, the HSCs are also expanded in thepresence of a fusion protein containing a protein of the presentdisclosure that inhibits apoptosis fused to a PTD (e.g., a TAT-Bcl-2fusion protein). For example, HSCs may be expanded by culturing thecells in the presence of a PTD-MYC and/or PTD-ICN-1 fusion protein, andoptionally in the presence of a PTD-Bcl-2 fusion protein and additionalcytokines and/or growth factors, for at least one day, at least twodays, at least three days, at least four days, at least five days, atleast six days, at least seven days, at least eight days, at least ninedays, at least 10 days, at least 11 days, at least 12 days, at least 13days, at least 14 days, or more.

Accordingly, HSCs suitable for use in any of the methods of the presentdisclosure may be obtained from embryonic stem cells (ES cells), fetalstem cells, induced pluripotent stem cells (iPS cells), bone marrow,from an apheresis procedure, from peripheral blood cells, fromperipheral blood cells that have undergone leukapheresis, from umbilicalcord blood, from amniotic fluid, from placenta, from cultured HSC cells,from an immortalized HSC cell line, or from a conditionally immortalizedHSC cell line.

Production of Red Blood Cells

Certain aspects of the present disclosure relate to methods forproducing a population of mature red blood cells from HSCs, by culturingthe HSCs in the presence of EPO, optionally IL-3, and one or morerecombinant protein of the present disclosure that promotes cellsurvival and/or proliferation under conditions that inducedifferentiation of the HSCs to mature red bloods cells, therebyproducing the population of mature red blood cells. The recombinantprotein may be exogenously provided or provided through transgenicmanipulation of the HSCs. Alternatively the protein that promotes cellsurvival and/or proliferation may be an endogenous protein that isinduced to be overexpressed. In some embodiments, the recombinant,induced, and/or exogenous protein is an onco-peptide of the presentdisclosure, MYC, ICN-1, homologues thereof, analogues thereof, and/orbiologically active fragments thereof. Optionally the protein thatpromotes cell survival and/or proliferation may form part of a fusionprotein. In some embodiments, the fusion protein includes one or more ofa PTD, an epitope tag, or a protein purification tag. In someembodiments, the HSCs are modified to include one or more proteins ofinterest before being differentiated into red blood cells. In someembodiments, conditional immortalization and/or inclusion of one or moreproteins of interest may be achieved through any method known in the artand described herein, such as one or more of a transgenic approach, aprotein-transduction approach, or an approach enhancing the expressionof endogenous proteins

In some embodiments of the methods of the present disclosure, acomposition containing a fusion protein containing a protein of thepresent disclosure that promotes cell survival and/or proliferationfused to a PTD (e.g., a PTD-MYC protein and/or PTD-ICN-1 protein fusion)is administered during the step of culturing the conditionallyimmortalized HSCs in the presence of EPO, and optionally IL-3 and othercomponents. In some embodiments, the HSCs are cultured in the presenceor in the absence of feeder cells and/or serum. In certain preferredembodiments, the HSCs are cultured in the absence of feeder cells and/orserum.

In some embodiments, the HSCs are cultured in the presence of at least0.5 μ/ml, at least 0.6 μ/ml, at least 0.7 μ/ml, at least 0.8 μml, atleast 0.9 μ/ml, at least 1 μ/ml, at least 2 μ/ml, at least 3 μ/ml, atleast 4 μ/ml, at least 5 μ/ml, at least 6 μ/ml, at least 7 μ/ml, atleast 8 μ/ml, at least 9 μ/ml, at least 10 μ/ml, at least 15 μ/ml, atleast 20 μ/ml, at least 25 μ/ml, at least 30 μ/ml, at least 35 μ/ml, atleast 40 μ/ml, at least 45 μ/ml, at least 50 μ/ml, at least 55 μ/ml, atleast 60 μ/ml, at least 65 μ/ml, at least 70 μ/ml, at least 75 μ/ml, atleast 80 μ/ml, at least 85 μ/ml, at least 90 μ/ml, at least 95 μ/ml, orat least 100 μ/ml of recombinant protein that promotes cell survivaland/or proliferation (e.g., MYC, ICN-1, homologues thereof, analoguesthereof, and/or biologically active fragments thereof).

In some embodiments, the HSCs are cultured in the presence of at least0.5 μ/ml, at least 0.6 μ/ml, at least 0.7 μ/ml, at least 0.8 μ/ml, atleast 0.9 μ/ml, at least 1 μ/ml, at least 2 μ/ml, at least 3 μ/ml, atleast 4 μ/ml, at least 5 μ/ml, at least 6 μ/ml, at least 7 μ/ml, atleast 8 μ/ml, at least 9 μ/ml, at least 10 μ/ml, at least 15 μ/ml, atleast 20 μ/ml, at least 25 μ/ml, at least 30 μ/ml, at least 35 μ/ml, atleast 40 μ/ml, at least 45 μ/ml, at least 50 μ/ml, at least 55 μ/ml, atleast 60 μ/ml, at least 65 μ/ml, at least 70 μ/ml, at least 75 μ/ml, atleast 80 μ/ml, at least 85 μ/ml, at least 90 μ/ml, at least 95 μ/ml, orat least 100 μ/ml MYC.

In certain embodiments, the HSCs, optionally conditionally immortalizedHSCs, are cultured in the presence of at least 0.5 μ/ml, at least 0.6μ/ml, at least 0.7 μ/ml, at least 0.8 μ/ml, at least 0.9 μ/ml, at least1 μ/ml, at least 2 μ/ml, at least 3 μ/ml, at least 4 μ/ml, at least 5μ/ml, at least 6 μ/ml, at least 7 μ/ml, at least 8 μ/ml, at least 9μ/ml, at least 10 μ/ml, at least 15 μ/ml, at least 20 μ/ml, at least 25μ/ml, at least 30 μ/ml, at least 35 μ/ml, at least 40 μ/ml, at least 45μ/ml, at least 50 μ/ml, at least 55 μ/ml, at least 60 μ/ml, at least 65μ/ml, at least 70 μ/ml, at least 75 μ/ml, at least 80 μ/ml, at least 85μ/ml, at least 90 μ/ml, at least 95 μ/ml, or at least 100 μ/ml ICN-1.

In certain embodiments, the HSCs, optionally conditionally immortalizedHSCs, are cultured in the presence of at least 1.0 unit/ml EPO, at least1.2 units/ml EPO, at least 1.4 units/ml EPO, at least 1.6 units/ml EPO,at least 1.8 units/ml EPO, at least 2.0 units/ml EPO, at least 2.2units/ml EPO, at least 2.4 units/ml EPO, at least 2.6 units/ml EPO, atleast 2.8 units/ml EPO, at least 3.0 units/ml EPO, at least 3.2 units/mlEPO, at least 3.4 units/ml EPO, at least 3.6 units/ml EPO, at least 3.8units/ml EPO, at least 4.0 units/ml EPO, or more EPO.

In certain embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of at least 1 ng/ml IL-3, atleast 2 ng/ml IL-3, at least 3 ng/ml IL-3, at least 4 ng/ml IL-3, atleast 5 ng/ml IL-3, at least 6 ng/ml IL-3, at least 7 ng/ml IL-3, atleast 8 ng/ml IL-3, at least 9 ng/ml IL-3, at least 10 ng/ml IL-3, atleast 11 ng/ml IL-3, at least 12 ng/ml IL-3, at least 13 ng/ml IL-3, atleast 14 ng/ml IL-3, at least 15 ng/ml IL-3, at least 16 ng/ml IL-3, atleast 17 ng/ml IL-3, at least 18 ng/ml IL-3, at least 19 ng/ml IL-3, atleast 20 ng/ml IL-3, at least 21 ng/ml IL-3, at least 22 ng/ml IL-3, atleast 23 ng/ml IL-3, at least 24 ng/ml IL-3, at least 25 ng/ml IL-3, ormore IL-3.

In certain embodiments, the HSCs, optionally conditionally immortalizedHSCs, are cultured in the presence of at least 1 ng/ml IL-3, at least 2ng/ml IL-3, at least 3 ng/ml IL-3, at least 4 ng/ml IL-3, at least 5ng/ml IL-3, at least 6 ng/ml IL-3, at least 7 ng/ml IL-3, at least 8ng/ml IL-3, at least 9 ng/ml IL-3, at least 10 ng/ml IL-3, at least 11ng/ml IL-3, at least 12 ng/ml IL-3, at least 13 ng/ml IL-3, at least 14ng/ml IL-3, at least 15 ng/ml IL-3, at least 16 ng/ml IL-3, at least 17ng/ml IL-3, at least 18 ng/ml IL-3, at least 19 ng/ml IL-3, at least 20ng/ml IL-3, at least 21 ng/ml IL-3, at least 22 ng/ml IL-3, at least 23ng/ml IL-3, at least 24 ng/ml IL-3, at least 25 ng/ml IL-3, or moreIL-3; and at least 1.0 unit/ml EPO, at least 1.2 units/ml EPO, at least1.4 units/ml EPO, at least 1.6 units/ml EPO, at least 1.8 units/ml EPO,at least 2.0 units/ml EPO, at least 2.2 units/ml EPO, at least 2.4units/ml EPO, at least 2.6 units/ml EPO, at least 2.8 units/ml EPO, atleast 3.0 units/ml EPO, at least 3.2 units/ml EPO, at least 3.4 units/mlEPO, at least 3.6 units/ml EPO, at least 3.8 units/ml EPO, at least 4.0units/ml EPO, or more EPO.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are cultured in the presence of EPO, and optionally IL-3, for atleast one day, at least two days, at least three days, at least fourdays, at least five days, at least six days, at least seven days, atleast eight days, at least nine days, at least 10 days, or longer.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of about 1-500 ng/ml FLT-3,about 1-500 ng/ml SCF, about 1-500 ng/ml GM-CSF, and/or about 1-500ng/ml TPO. The HSCs, optionally conditionally immortalized HSCs, may befurther cultured in the presence of FLT-3, SCF, GM-CSF, and/or TPO forat least one day, at least two days, at least three days, at least fourdays, at least five days, at least six days, at least seven days, atleast eight days, at least nine days, at least 10 days, or longer. TheFLT-3, SCF, GM-CSF, and/or TPO may be added to the culture media at anypoint during the period of time when the HSCs are differentiating intomature red blood cells, and/or after the mature red blood cells havebeen produced.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of at least 1 ng/ml FLT-3, atleast 2 ng/ml FLT-3, at least 3 ng/ml FLT-3, at least 4 ng/ml FLT-3, atleast 5 ng/ml FLT-3, at least 6 ng/ml FLT-3, at least 7 ng/ml FLT-3, atleast 8 ng/ml FLT-3, at least 9 ng/ml FLT-3, at least 10 ng/ml FLT-3, atleast 15 ng/ml FLT-3, at least 20 ng/ml FLT-3, at least 25 ng/ml FLT-3,at least 30 ng/ml FLT-3, at least 35 ng/ml FLT-3, at least 40 ng/mlFLT-3, at least 45 ng/ml FLT-3, at least 50 ng/ml FLT-3, at least 55ng/ml FLT-3, at least 60 ng/ml FLT-3, at least 65 ng/ml FLT-3, at least70 ng/ml FLT-3, at least 75 ng/ml FLT-3, at least 80 ng/ml FLT-3, atleast 85 ng/ml FLT-3, at least 90 ng/ml FLT-3, at least 95 ng/ml FLT-3,at least 100 ng/ml FLT-3, at least 150 ng/ml FLT-3, at least 200 ng/mlFLT-3, at least 250 ng/ml FLT-3, at least 300 ng/ml FLT-3, at least 350ng/ml FLT-3, at least 400 ng/ml FLT-3, at least 450 ng/ml FLT-3, atleast 500 ng/ml FLT-3, or more of FLT-3.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of at least 1 ng/ml SCF, atleast 2 ng/ml SCF, at least 3 ng/ml SCF, at least 4 ng/ml SCF, at least5 ng/ml SCF, at least 6 ng/ml SCF, at least 7 ng/ml SCF, at least 8ng/ml SCF, at least 9 ng/ml SCF, at least 10 ng/ml SCF, at least 15ng/ml SCF, at least 20 ng/ml SCF, at least 25 ng/ml SCF, at least 30ng/ml SCF, at least 35 ng/ml SCF, at least 40 ng/ml SCF, at least 45ng/ml SCF, at least 50 ng/ml SCF, at least 55 ng/ml SCF, at least 60ng/ml SCF, at least 65 ng/ml SCF, at least 70 ng/ml SCF, at least 75ng/ml SCF, at least 80 ng/ml SCF, at least 85 ng/ml SCF, at least 90ng/ml SCF, at least 95 ng/ml SCF, at least 100 ng/ml SCF, at least 150ng/ml SCF, at least 200 ng/ml SCF, at least 250 ng/ml SCF, at least 300ng/ml SCF, at least 350 ng/ml SCF, at least 400 ng/ml SCF, at least 450ng/ml SCF, at least 500 ng/ml SCF, or more of SCF.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of at least 1 ng/ml GM-CSF,at least 2 ng/ml GM-CSF, at least 3 ng/ml GM-CSF, at least 4 ng/mlGM-CSF, at least 5 ng/ml GM-CSF, at least 6 ng/ml GM-CSF, at least 7ng/ml GM-CSF, at least 8 ng/ml GM-CSF, at least 9 ng/ml GM-CSF, at least10 ng/ml GM-CSF, at least 15 ng/ml GM-CSF, at least 20 ng/ml GM-CSF, atleast 25 ng/ml GM-CSF, at least 30 ng/ml GM-CSF, at least 35 ng/mlGM-CSF, at least 40 ng/ml GM-CSF, at least 45 ng/ml GM-CSF, at least 50ng/ml GM-CSF, at least 55 ng/ml GM-CSF, at least 60 ng/ml GM-CSF, atleast 65 ng/ml GM-CSF, at least 70 ng/ml GM-CSF, at least 75 ng/mlGM-CSF, at least 80 ng/ml GM-CSF, at least 85 ng/ml GM-CSF, at least 90ng/ml GM-CSF, at least 95 ng/ml GM-CSF, at least 100 ng/ml GM-CSF, atleast 150 ng/ml GM-CSF, at least 200 ng/ml GM-CSF, at least 250 ng/mlGM-CSF, at least 300 ng/ml GM-CSF, at least 350 ng/ml GM-CSF, at least400 ng/ml GM-CSF, at least 450 ng/ml GM-CSF, at least 500 ng/ml GM-CSF,or more of GM-CSF.

In further embodiments, the HSCs, optionally conditionally immortalizedHSCs, are further cultured in the presence of at least 1 ng/ml TPO, atleast 2 ng/ml TPO, at least 3 ng/ml TPO, at least 4 ng/ml TPO, at least5 ng/ml TPO, at least 6 ng/ml TPO, at least 7 ng/ml TPO, at least 8ng/ml TPO, at least 9 ng/ml TPO, at least 10 ng/ml TPO, at least 15ng/ml TPO, at least 20 ng/ml TPO, at least 25 ng/ml TPO, at least 30ng/ml TPO, at least 35 ng/ml TPO, at least 40 ng/ml TPO, at least 45ng/ml TPO, at least 50 ng/ml TPO, at least 55 ng/ml TPO, at least 60ng/ml TPO, at least 65 ng/ml TPO, at least 70 ng/ml TPO, at least 75ng/ml TPO, at least 80 ng/ml TPO, at least 85 ng/ml TPO, at least 90ng/ml TPO, at least 95 ng/ml TPO, at least 100 ng/ml TPO, at least 150ng/ml TPO, at least 200 ng/ml TPO, at least 250 ng/ml TPO, at least 300ng/ml TPO, at least 350 ng/ml TPO, at least 400 ng/ml TPO, at least 450ng/ml TPO, at least 500 ng/ml TPO, or more of TPO.

Current methods of producing red blood cells from primary HSCs, forexample see Giarratana et al., (2005) Nat Biotech 23, 69-74, require atleast three weeks (21 days) to produce red blood cells. However, themethods of the present disclosure for producing a population of redblood cells from conditionally immortalized HSCs produce red blood cellsin about 10 days. As compared to the at least 21 days of the currentmethods, the 10 days of the presently disclosed methods represents anacceleration of approximately 52%. Thus, in some embodiments, theproduction of the population of mature red blood cells is accelerated byat least 45%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or more compared to production of a populationof red blood cells from a primary stem cell cultured in the presence ofIL-3 and EPO for eight days, then in the presence of feeder cells andEPO for three days, and finally in the presence of feeder cells alonefor 10 days (see, Giarratana et al., (2005) Nat Biotech 23, 69-74).

In other embodiments, the population of mature red blood cells isproduced in about 7 to 14 days. In other embodiments, the population ofmature red blood cells is produced in about 3 days, about 4, about 5days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, about 12 days, about 13 days, or about 14 days.

In other embodiments of the methods of the present disclosure, theproduced population of red blood cells is a population of fully maturered blood cells. In still other embodiments, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100% of the cells in the population of mature red blood cells areanucleated. In yet other embodiments, the population of mature red bloodcells is continually produced from conditionally immortalized HSCs.

In other embodiments of the methods of the present disclosure, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100% of the cells in the population ofmature red blood cells express Glycophrin A (GPA). In furtherembodiments, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% of the cells inthe population of mature red blood cells exhibit decreased levels ofCD71 (transferrin receptor) expression. In further embodiments, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100% of the cells in the population ofmature red blood cells exhibit decreased levels of fetal hemoglobinexpression. In further embodiments, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% of the cells in the population of mature red blood cells expressadult hemoglobin.

In other embodiments, the produced population of mature red blood cellsis a population of human cells. In further embodiments, the producedpopulation of mature red blood cells is a population of non-human animalcells, including, without limitation, a population of cells fromlaboratory/research animals, rodents, pets, livestock, farm animals,work animals, pack animals, rare or endangered species, racing animals,zoo animals, monkies, primates, mice, rats, guinea pigs, hamsters, dogs,cats, horses, cows, pigs, sheep, goats, and chickens.

The methods of the present disclosure may also utilize HSCs, optionallyconditionally immortalized HSCs, derived from human HSCs obtained fromdonors having a blood of type A+, A−, B+, B−, O+, O−, AB+, or AB−.Accordingly, the red blood cells produced from the HSCs will be of bloodtype A+, A−, B+, B−, O+, O−, AB+, or AB−.

Additionally, the methods of the present disclosure may also utilizeHSCs, optionally conditionally immortalized HSCs derived from human HSCsobtained from donors having a rare blood type including, withoutlimitation, Oh, CDE/CDE, CdE/CdE, C^(w)D−/C^(w)D−, −D−/−D−, Rh_(null),Rh: −51, LW(a−b+), LW(a−b−), S−s−U−, S−s−U(+), pp, Pk, Lu(a+b−),Lu(a−b−), Kp(a+b−), Kp(a−b−), Js(a+b−), Ko, K: −11, Fy(a−b−), Jk(a−b−),Di(b−), I−, Yt(a−), Sc: −1, Co(a−), Co(a−b−), Do(a−), Vel−, Ge−, Lan−,Lan(+), Gy(a−), Hy−, At(a−), Jr(a−), In(b−), Tc(a−), Cr(a−), Er(a−),Ok(a−), JMH−, and En(a−). Accordingly, in some embodiments, thepopulation of red blood cells produced from the HSCs will be of a rareblood type including, without limitation, Oh, CDE/CDE, CdE/CdE,C^(W)D−/C^(W)D−, −D−/−D−, Rh_(null), Rh: −51, LW(a−b+), LW(a−b−),S−s−U−, S−s−U(+), pp, Pk, Lu(a+b−), Lu(a−b−), Kp(a+b−), Kp(a−b−),Js(a+b−), Ko, K: −11, Fy(a−b−), Jk(a−b−), Di(b−), I−, Yt(a−), Sc: −1,Co(a−), Co(a−b−), Do(a−), Vel−, Ge−, Lan−, Lan(+), Gy(a−), Hy−, At(a−),Jr(a−), In(b−), Tc(a−), Cr(a−), Er(a−), Ok(a−), JMH−, and En(a−).

Additionally, the methods of the present disclosure may also utilizeHSCs, optionally conditionally immortalized HSCs derived from donorshaving an auto-immune disorder, immune deficiency, or any other diseaseor disorder that would benefit from a transplantation of HSCs and/ortransfusion of blood to produce a population of mature red blood cellsthat can be used for personalized therapies. For example, the populationof mature red blood cells may be produced from HSCs obtained from anautologous or allogenic donor. Advantageously, autologous red bloodcells may be particularly useful in cases where the subject in need of ablood transfusion and/or treatment with red blood cells would have anegative, deleterious, or toxic reaction to treatment with red bloodcells derived or obtained from any other donor, including allogenicand/or universal donors. Examples of patients that may benefit fromtreatment with red blood cells produced from HSCs derived fromautologous and/or allogenic donors are well known in the art andinclude, without limitation, those suffering from an autoimmunedisorder, blood disease or disorder, immune disease or disorder, orother related diseases or conditions.

In certain embodiments the population of red blood cells is producedfrom conditionally immortalized HSCs that can be passaged indefinitelyin vitro, cryopreserved, and recovered. Accordingly, such conditionallyimmortalized HSCs allow for the continuous production of fullydifferentiated red blood cells from a defined, well-characterized,source.

The methods of the present disclosure may utilize HSCs from any source,including but not limited to, primary hematopoietic stem cells from cordblood, placenta, peripheral blood, bone marrow, or mobilized blood.Hematopoietic stem cells derived from embryonic stem cells, fetal bloodcells or induced pluripotent stem cells, as well as conditionallyimmortalized hematopoietic stem cells such as transgenic and proteintransduced conditionally immortalized cells are expressly contemplated.

Populations of Red Blood Cells

Certain aspects of the present disclosure relate to populations of redblood cells, such as mature red blood cells, optionally produced by oneor more methods of the present disclosure. Populations of red bloodcells may also include one or more proteins of interest of the presentdisclosure. These proteins of interest may be useful in prevention,treatment, and/or diagnosis of one or more diseases or disorders asdisclosed herein.

In some aspects, populations of red blood cell may be characterized byone or more characteristics, including but not limited to, at leastabout 40%, at least 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, or about 100% of the cells in thepopulation are anucleated, express GPA, express adult hemoglobin (seconddecade or higher by FACS), exhibit decreased levels of CD71 expression(e.g. GPA⁺/CD71⁻), and/or exhibit decreased levels of fetal hemoglobin(first decade; 0-10 by FACS). Populations of red blood cells may alsoinclude one or more recombinant proteins of interest. These proteins ofinterest may be useful in prevention, treatment, and/or diagnosis of oneor more diseases or disorders as disclosed herein.

In some embodiments, a population of red blood cells may further includean exogenous protein of the present disclosure that inhibits apoptosis,such as a protein containing a Bcl-2 homology domain. The exogenousprotein may also be a fusion protein that contains a PTD. In someembodiments, the exogenous protein is Bcl-2, a homologue thereof, ananalogue thereof, and/or a biologically active fragment thereof. Incertain embodiments, the exogenous protein is Bcl-2, optionallyPTD-Bcl-2. In some embodiments, the population of red blood cells aremaintained in storage media that includes an protein of the presentdisclosure that inhibits apoptosis, such as a protein containing a Bcl-2homology domain. In some embodiments, the exogenous protein in thestorage media is Bcl-2, a homologue thereof, an analogue thereof, and/ora biologically active fragment thereof. In certain embodiments, theexogenous protein in the storage media is Bcl-2, optionally PTD-Bcl-2Bcl-2.

Pharmaceutical Compositions

Certain aspects of the present disclosure relate to pharmaceuticalcompositions including one or more populations of red blood cells of thepresent disclosure and one or more pharmaceutically acceptableexcipients. Any pharmaceutically acceptable excipient known in the artthat is suitable for use with red blood cells may be used. In onenon-limiting example, the pharmaceutically acceptable excipient is apH-balanced saline solution.

In some embodiments, the composition further includes one or moreproteins of interest of the present disclosure.

The populations of red bloods are optionally produced by one or moremethods of the present disclosure and/or exhibit one or more ofcharacteristics of the present disclosure, including without limitation,about 40% to about 100% of the red blood cells in the population areanucleated, about 40% to about 100% of the red blood cells in thepopulation express adult hemoglobin, about 40% to about 100% of the redblood cells in the population exhibit increased expression of adulthemoglobin, about 40% to about 100% of the red blood cells in thepopulation exhibit decreased levels of CD71 expression, and about 40% toabout 100% of the red blood cells in the population exhibit decreasedlevels of fetal hemoglobin expression. Additionally, the population ofred blood cells may have a rare blood type. In some embodiments, the redblood cells are human red blood cells. Alternatively, the red bloodcells are non-human red blood cells derived from any non-human animal ofthe present disclosure.

The populations of red blood cells may also include one or more proteinof interest of the present disclosure. In some embodiments, the one ormore proteins of interest are associated on the surface of the red bloodcells.

Pharmaceutical compositions of the present disclosure containing a oneor more populations of red blood cells of the present disclosure and oneor more pharmaceutically acceptable excipients, where the red bloodcells optionally contain one or more proteins of interest of the presentdisclosure may be formulated for in vivo administration, such as throughtransfusion. Any formulation known in the art for in vivo administrationof a pharmaceutical composition containing a population of red bloodcells may be used.

Therapeutic Uses

Red blood cells described herein and/or produced according to any of themethods of the present disclosure for producing a population of maturered blood cells from HSCs, optionally conditionally immortalized HSCsalso find use in therapeutic applications.

The use of red blood cell transfusions for patients in need of suchtreatment for a variety of reasons and disorders is well known in theart, and approaches are standard medical practice. The red blood cellsdescribed herein and/or produced according to the methods of the presentdisclosure can be used to treat patients using the same approaches andconditions currently used for blood transfusions.

In certain embodiments, the present disclosure relates to methods oftreatment, prevention, or diagnosis of a disease or disordercharacterized by a deficiency of red blood cells by administering apopulation of red blood cells of the present disclosure, or preparedaccording to any of the methods of the present disclosure, to a subjecthaving a disorder characterized by a deficiency of red blood cells. Insome embodiments, about 40% to about 100% of the red blood cells in thepopulation are anucleated, about 40% to about 100% of the red bloodcells in the population express adult hemoglobin, about 40% to about100% of the red blood cells in the population exhibit increasedexpression of adult hemoglobin, about 40% to about 100% of the red bloodcells in the population exhibit decreased levels of CD71 expression, andabout 40% to about 100% of the red blood cells in the population exhibitdecreased levels of fetal hemoglobin expression.

As used herein, a “deficiency of red blood cells,” refers to a subjectthat has an amount of red blood cells that is from about 20% to about900% lower than the amount of red blood cells in a subject having anormal amount of red blood cells; or has an amount of red blood cellsthat is from about 10 times to about 1,000 times lower than the amountof red blood cells in a subject having a normal amount of red bloodcells.

Disorders characterized by a deficiency of red blood cells may include,without limitation, anemia (e.g., congenital anemia, aplastic anemia,pernicious anemia, iron deficiency anemia, sickle cell anemia,spherocytosis, hemolytic anemia, Aceruloplasminemia, Adenosine deaminaseincreased activity—ADA—, Adenylate kinase deficiency, Aldolasedeficiency, Alpha-thalassaemia—trait or carrier, Atransferrinemia,Autosomal dominant sideroblastic anemia, Autosomal recessivesideroblastic anemia, Beta-thalassaemia—trait or carrier,Beta-thalassaemia major (and intermedia), CDA with thrombocytopenia(GATA I mutation), Compound heterozygous sickling disorders, Congenitalacanthocytosis, Congenital dyserythropoietic anaemia type I, Congenitaldyserythropoietic anaemia type II, Congenital dyserythropoietic anaemiatype III, Delta Beta-thalassaemia, Diamond-Blackfan-Anemia,DMT1-deficiency anaemia, Familial hypoplastic anaemia, Fanconi anaemia,Gamma-glutamyl-cysteine synthetase deficiency, GLRX5-relatedSideroblastic anaemia, Glucose phosphate isomerase deficiency,Glucose-6-phosphate dehydrogenase deficiency, Glutathione reductasedeficiency, Glutathione synthetase deficiency, Haemoglobin C disease,Haemoglobin D disease, Haemoglobin E disease, Haemoglobin H disease,Haemoglobin Lepore, Haemoglobin M with anaemia, HereditaryElliptocytosis, Hereditary persistance of fetal haemoglobin, HereditarySpherocytosis, Hereditary Stomatocytosis, Hexokinase deficiency, Hydropsfetalis, Imerslund-Gräsbeck-Syndrom, Iron—refractory iron deficiencyanemia, Kearns-Sayre syndrome, Lecithin cholesterol acyltransferasedeficiency, Mitochondrial myopathy sideroblastic anemia, thalassaemias,congenitale dyserythropoetic anemia, Pancytopenia with malformations,Paroxysmal nocturnal hemoglobinuria, Pearson's Syndrome,Phosphofructokinase deficiency, Phosphoglycerate kinase deficiency,Pyrimidine 5 nucleotidase deficiency, Pyruvate kinase deficiency, Sicklecell anemia, Sickle cell trait, Sideroblastic anemia associated withataxia, SLC25A38-related Sideroblastic anemia, Thiamine-responsivemegaloblastic anemia, Triose phosphate isomerase deficiency, Unstablehaemoglobin, Wolfram Syndrome, and X-linked sideroblastic anemia),Gaucher's disease, hemolysis, neutropenia, thrombocytopenia,granulocytopenia, hemophilia, Hodgkin's lymphoma, Non-Hodgkin'slymphoma, B cell chronic lymphoma, Burkitt's lymphoma, Follicular-likelymphoma, diffused large B-cell lymphoma, multiple myeloma, acutemyeloid leukemia, pre-B acute lymphocytic leukemia, pre-T acutelymphocytic leukemia, acute promyelocytic leukemia, refractory leukemia,or combinations thereof.

In certain instances, the disorder characterized by a deficiency of redblood cells results from (partially or fully) one or more ofchemotherapy, chemical exposure, radiation therapy, and/or radiationexposure. In some embodiments, a population of red blood cells producedby any method of the present disclosure is co-administered withchemotherapy and/or radiation therapy or one or more protein ofinterest.

In some embodiments, a population of red blood cells of the presentdisclosure, and/or produced according to any method of the presentdisclosure is administered to or transfused into a cells subject in needthereof, e.g., suffering from a loss of blood. A loss of blood may bethe result of for example, internal or external bleeding, hemorrhage,trauma, or surgery, among others.

Treatment with one or more of the population of red blood cells of thepresent disclosure may also be useful for some infectious diseasesassociated with hemorrhage, such as but not limited to, families of RNAviruses (Arenaviridae, Bunyaviridae, Filoviridae, and Flaviviridae) thatare linked to viral hemorrhagic fever. Examples of viral hemorrhagicfevers including but are not limited to, Lassa fever, Ebola, Marburg,Rift Valley fever, dengue, and yellow fever.

In embodiments where an immediate transfusion is needed, largequantities of red blood cells described herein and/or prepared accordingto any method of the present disclosure are administered to anindividual. In other embodiments, sustained transfusion of the producedpopulation of red blood cells is administered to the individual.

For treatment of some diseases, disorders, and/or conditions it isuseful to administer red blood cells adapted to be a delivery system forone or more proteins of interest of the present disclosure. Any methodsof adapting red blood cells to be a delivery system for proteins knownin the art and disclosed herein may be used. Any disease disorder and/orcondition known in the art and disclosed herein that would benefit fromtreatment with a disclosed protein of interest may be treated with themethods of the present disclosure, including, without limitation,subjects in need of hematopoietic growth factors, acute inflammatoryconditions, cytokine storm conditions, clinical signs associate withcytokine storms, cancer, vascular dysregulation (e.g., frost bite,cancer-related vasoconstriction, or rheumatic joints, etc), acutecardiac infarctions, obstetrical uses during child delivery, acuteand/or persistent migraine headaches, subjects in need of an immunitybooster, subjects at high risk of having clots, subjects at elevatedrisk for pulmonary embolisms, cardiovascular diseases, immune diseasesand/or disorder, and autoimmune diseases and/or disorders.

A “subject”, “patient”, or “host” to be treated by any of the methods ofthe present disclosure may be any human or non-human animal, such as anyof the non-human animal disclosed herein, in need of such treatment. Forexample, the subject may have a deficiency of red blood cells, has anautoimmune deficiency, an anemia, cancer, or any other disease,disorder, or condition known in the art and disclosed herein that may betreated by red blood cells of the present disclosure and/or one or moreproteins of interest of the present disclosure.

Citation of documents and studies referenced herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the contents of these documents.

The following Examples are merely illustrative and are not meant tolimit any aspects of the present disclosure in any way.

EXAMPLES

The following examples describe the results of differentiating murineand human conditionally transformed long-term hematopoietic stem celllines (clt-HSC) and protein transduced long-term hematopoietic stemcells (ptlt-HSC) to red blood cells (RBCs) in vitro.

The described approach facilitates the rapid production of populationsof mature enucleated red blood cells that are optionally payloaded withone or more proteins of interest. In addition, generation ofconditionally immortalized HSCs as the starting material allows forproduction and maintenance of universal donor blood, or a variety ofmajor blood types as well as rare blood types and even personalizedblood to resolve the problem of continuous blood shortage that patientswith rare blood types have encountered over the years. Further, thistechnology can be used provide a constant, predictable supply of RBCsfor transfusions that are derived from a defined source that can becertified to be pathogen-free and have an extended shelf life that willallow the establishment of a sufficient stock supply of RBCs.

Example 1: Generation of Conditionally Transformed Long-TermReconstituting Hematopoietic Stem Cell Lines

HSC-enriched bone marrow (BM) cells were prepared by treatment of micewith 5-fluorouracil (5FU) to ablate proliferating cells. Ex vivo BMcells from treated mice were further enriched for HSCs by culturing inmedium containing IL-3, IL-6, and SCF, as previously described (VanParijs et al., Immunity 11, 763-770, 1999). Cells were then subjected tothree rounds of spin infection with pMIG-MYC-ER and pMIG-Bcl-2 virusesencoding oncoproteins as well as green fluorescent protein (FIG. 1A)(Refaeli et al., J. Exp. Med. 196, 999-1005, 2002). Variants of thepMSCV backbone were generated to encode the cDNAs for human MYC-ER orBcl-2 as well as an IRES element and a reporter gene (EGFP). Theresulting viruses generated bi-cistronic transcripts such that the levelof reporter gene expression correlated with the level of expression ofthe first cDNA.

This treatment yielded a rate of retroviral transduction ofapproximately 33.7%, as determined by the frequency of green fluorescentprotein (GFP) expressing cells 96 hours after the initial cultures wereestablished (FIG. 1B). 10⁵ transduced HSCs were transplanted intocohorts of young, lethally irradiated, male C57/BL6 mice, and these micewere given weekly injections of 4-hydroxytamoxifen (4-OHT) in an amountof 1 mg/mouse/week, beginning 10 days after transplantation. Leukemiasdeveloped in over 90% of these mice with a consistent latency period offour weeks (FIG. 1C). The curve in FIG. 1C represents the percentage ofsurviving mice at a given point in time after the 4-OHT injections began(day 0 in the graph). All of the mice died uniformly from an AML-likeleukemia after about 40 days (FIG. 1C). The data shown in FIG. 1 werefrom one experiment representative of 4 independent experiments.Development of leukemias required the continuous administration of4-OHT.

Although the specific example provided is directed to the conditionalimmortalization of HSCs using Myc and Bcl-2, a similar approach would beused to incorporate other proteins of interest into HSCs, and optionallyto control the expression of the proteins of interest. In someembodiments, the HSCs would include conditionally immortalized HSCs orprotein transduced HSCs.

Example 2: Conditionally Transformed HSC Cell Lines Exhibiting a Lt-HSCSurface Phenotype

To assess the phenotype and homogeneity of the cell lines developed inExample 1, the cellular expression of a variety of surface markers wasanalyzed. Cells were stained with antibodies to c-kit, Sca-1, CD34, andFlk-2. Additionally, the cells were stained for specific lineages: CD19and B220 for B-lineage cells, Thy1.2 for T-lineage cells, Mac-1 formyeloid cells, Gr-1 for neutrophils, and Ter-119 for red blood cellprogenitors. As shown in Figures. 1D-1F, the phenotype consistentlyobserved was lineage negative (CD19−, B220−), but Sca-1⁺, c-kit⁺, CD34⁻,and Flk-2⁻. This pattern of marker expression is consistent with thatpreviously reported for murine primary Lt-HSCs (Cheshier et al., Proc.Natl Acad. Sci. USA 96, 3120-3125, 1999). It was also noted that whenctlt-HSC cell lines were maintained in culture for extended periods oftime, cellular c-kit expression decreased (FIG. 1E). This change is notassociated with detectable changes in in vivo or in vitro function.Moreover, it was also found that c-Kit levels were restored when cellswere cultured overnight without SCF, suggesting that SCF drivesmodulation of its receptor.

In particular, FIG. 1D shows ctlt-HSCs obtained from the bone marrow ofleukemic mice soon after recovery. In these samples, approximately 42.2%of the cells were Sca-1⁺ and c-kit⁺; approximately 100% of the cellswere CD34⁻ and Flk-2⁻; and approximately 99.8% of the cells were B220⁻and CD19⁻ (FIG. 1D).

FIG. 1E shows ctlt-HSC cell lines that were maintained in culture forextended periods of time. In these samples, approximately 7.79% of thecells were Sca-1⁺ and c-kit⁺; approximately 51.2% of the cells wereSca-1⁺ but c-kit⁻; approximately 100% of the cells were CD34⁻ andFlk-2⁻; and approximately 99.4% of the cells were B220⁻ and CD19⁻ (FIG.1E).

FIG. 1F shows normal, unmanipulated Lt-HSCs obtained from the bonemarrow of wild type C57/BL6 mice. In these samples, only approximately1.84% of the cells were Sca-1⁺ and c-kit⁺, approximately 8.03% of thecells were Sca-1⁻ and c-kit⁺, while the majority of the cells(approximately 88.5%) were negative for both Sca-1 and c-kit (FIG. 1F).Additionally, while approximately 100% of the cells were CD34⁻, not allof the cells were Flk-2⁻ (FIG. 1F).

Moreover, as shown in FIG. 1D, at an early stage of the process ofestablishing a ctlt-HSC cell line, the cells predominantly express highlevels of c-kit and Sca-1, and do not express Flk-2, CD34, or lineagemarkers such as CD19 and B220. FACS analysis of an established ctlt-HSCcell line shows that once the ctlt-HSC cell line was expanded andcryopreserved, it retained a stable surface phenotype. These cellsexpressed high levels of Sca-1, but had reduced surface levels of c-Kit,and remained negative for Flk-2, CD34, B220, CD 19, and other lineagemarkers (FIG. 1E). The reduction of c-kit levels from the surfaceappears to be a result of continuous signaling, since they require SCFto retain their HSC-like phenotype. The results of the ctlt-HSC cellline were compared to normal, unmanipulated long-term HSCs from the bonemarrow of wild-type C57/BL6 mice. As shown in FIG. 1F, the cells werestained with antibodies to c-kit, sca-1, Flk-2, and CD34, in order tocompare the expression levels of the marker proteins from normal HSCsand from the ctlt-HSC cell lines.

Example 3: Rescue of Mice from Lethal Irradiation by Transplantation ofCtlt-HSCs

This example demonstrates that ability of ctlt-HSC cell lines to giverise to differentiated red blood cells (RBCs). This ability, as well ascompetence to maintain an active HSC compartment for extended periods oftime, is critical to establish the identity of ctlt-HSC as Lt-HSC. Theability of the ctlt-HSC cell lines to reconstitute the hematopoieticcompartment of lethal irradiated animals was examined in two ways.First, 10³ ctlt-HSC cells along were transferred with 3×10⁵ whole bonemarrow cells from Rag-1^(−/−) mice into lethally irradiated youngC57/BL6 mice. The addition of the “carrier” Rag-1^(−/−) cells ensuredthat recipients could produce red blood cells during the period of timerequired for transferred HSCs to re-establish erythropoiesis.Supplementation with whole “carrier” bone marrow cells is generally usedin conjunction with HSC reconstitution in order to allow transplantrecipients to survive the loss of existing red blood cells that followsirradiation (Uchida et al., J. Exp. Med. 175, 175-184, 1992). Thus, theexperiments were designed so that the only possible source of maturelymphoid cells was the transplanted ctlt-HSC cells. In a variation ofthis approach 10³ ctlt-HSC cells were transplanted into sublethallyirradiated Rag-1^(−/−) mice without carrier bone marrow. Mice receivingctlt-HSC were euthanized 6 or 12 weeks after transplantation and lymphnode, spleen, thymus, and bone marrow tissues were harvested foranalysis of reconstitution. The resulting cell suspensions were stainedwith lineage specific antibodies to ascertain the extent of thereconstitution. The cells that developed from ctlt-HSCs were traced invivo by virtue of the retrovirally encoded reporter gene, GFP.

The ctlt-HSC cell lines gave rise to bone marrow, thymus, spleen, andlymph node tissue with a high frequency of GFP⁺ cells (70-80% of viablerecovered cells) in bone marrow, thymus, spleen, and lymph nodes (FIG.2A). These histograms were derived from the organs in one mouseexemplary of each within a cohort of five. In particular, approximately78% of bone marrow cells were GFP+, approximately 72% of thymus cellswere GFP+, approximately 80% of spleen cells were GFP+, andapproximately 79% of lymph node (LN) cells were GFP+(FIG. 2A).

As shown in FIG. 2B, cells obtained from bone marrow were stained forMac-1 and Gr-1. While not all of the myeloid cells found in the bonemarrow expressed GFP, a significant portion was GFP⁺ and hence wasderived from the ctlt-HSCs. In particular, approximately 14.8% of thecells were Mac-1⁺, with approximately 24% of the cells being both GFP⁺and Mac-1⁺. Thus, approximately 61.8% of Mac-1⁺ cells were derived fromctlt-HSCs (FIG. 2B). Additionally, approximately 32% of the cells wereGr-1⁺, with approximately 18.7% of the cells being both GFP⁺ and Gr-1⁺.Thus, approximately 58.4% of Mac-1⁺ cells were derived from ctlt-HSCs(FIG. 2B).

As shown in FIG. 2C, cells obtained from spleen tissue of chimericRag-1^(−/−) mice were analyzed by flow cytometry for the presence ofmature T and B cells. Rag-1^(−/−) mice and wild type mice were used ascontrols. The cells were stained for the presence of TCRαβ T cells thatwere either CD4 or CD8 single positive. In addition, cells were stainedfor the presence of CD 19⁺ B cells that expressed IgM and IgD on theirsurface.

In particular, spleen cell samples from the chimeric Rag-1^(−/−) micehad approximately 25.8% TCRαβ⁺ and CD4⁺ cells, approximately 16.8%TCRαβ⁺ and CD8⁺ cells, and approximately 7.8% IgM⁺ and CD19⁺ cells (FIG.2C). This is compared to spleen cell samples from control Rag-1^(−/−)mice that had no TCRαβ⁺ and CD4⁺ cells, TCRαβ⁺ and CD8⁺ cells, or IgM⁺and CD 19⁺ cells; and spleen cell samples from wild type mice that hadapproximately 24% TCRαβ⁺ and CD4⁺ cells, approximately 16.1% TCRαβ⁺ andCD8⁺ cells, and approximately 28.7% IgM⁺ and CD19⁺ cells (FIG. 2C).

As can be seen in FIG. 2C, while the frequency of mature T-cells in thespleen was comparable to what can be found in wild-type, unmanipulatedC57/BL6 mice, the development of B-cells was delayed.

It was then determined whether the ctlt-HSCs were capable ofself-renewal following transplantation. Bone marrow cells obtained fromthe first set of ctlt-HSC transplant recipient mice were seriallytransplanted into a second cohort of lethally irradiated Rag-1^(−/−)mice. Reconstitution was analyzed 6 or 12 weeks later.

As shown in FIG. 2D, the secondary transplantation was also able to giverise to mature lineages. Spleens were collected from the cohort of thechimeric mice, and single cell suspensions were prepared and used forFACS analysis. The frequency of mature T and B cells found inrecipients, control Rag-1^(−/−) mice, and control wild-type C57/BL6 micewere compared. The analysis showed the presence of mature CD4 and CD8single positive TCRαβ T cells in the spleens at a frequency similar tothat of the wild-type mice. CD19⁺, IgM⁺, and IgD⁺ B cells were alsopresent, albeit at a lower frequency than in the wild-type mice.

In particular, spleen cell samples from the secondary transplantrecipient Rag-1^(−/−) mice had approximately 41.3% TCRαβ⁺ and CD4⁺cells, approximately 34.3% TCRαβ⁺ and CD8⁺ cells, and approximately10.2% IgM⁺ and CD19⁺ cells (FIG. 2D). This is compared to spleen cellsamples from control Rag-1^(−/−) mice that had approximately 0.7% TCRαβ⁺and CD4⁺ cells, approximately 0.4% TCRαβ⁺ and CD8⁺ cells, and 0.5% IgM⁺and CD19⁺ cells; and spleen cell samples from wild type mice that hadapproximately 36.9% TCRαβ⁺ and CD4⁺ cells, approximately 37.7% TCRαβ⁺and CD8⁺ cells, and approximately 16.9% IgM⁺ and CD19⁺ cells (FIG. 2D).

Seven successive serial transplants were subsequently performed andreconstitution of mature lineages from the initial 10³ ctlt-HSCs wasobserved with no evidence of tumor formation (Table 1). For thisexperiment, one million whole bone marrow cells were transplanted intolethally irradiated Rag-1^(−/−) mice. Transplantation into Rag1^(−/−)mice (Jackson Laboratory) was carried out as described for NSG miceexcept Rag1^(−/−) mice received two subsequent doses (2-3 hours apart)of 450 rads of radiation just prior to injection the BM cells via thetail vein.

TABLE 1 # repeat PB PB Transplant mice Avg T % Std Dev Avg B % Std Dev 18 29.300 14.106 15.190 12.834 2 9 48.910 17.396 15.230 16.619 3 4 36.9007.230 6.955 5.206 4 3 21.560 9.415 7.683 4.657 5 4 32.930 13.968 7.1401.373 6 7 16.051 5.582 0.7629 0.472 7 5 15.174 8.99 0.239 0.156 8 6 0.80.341 0.044 0.044

Example 4: Development of Human ctlt-HSC Cell Lines and Chimeric MiceBearing a Human Hematopoietic Compartment

A method for conditionally immortalizing murine long-term HSCs has beenpreviously developed. This approach was extended to conditionallyimmortalize human long-term HSCs so as to determine whether themechanisms responsible for conditionally immortalizing murine HSCs areuniversally applicable to any HSCs or whether they are specific only tomurine cells. In order to test this notion, the CD34⁺ fraction of humanadult bone marrow, or cord blood were obtained (initially from Stem CellTechnologies, Vancouver, BC, and then from the UCHSC cord blood bank).The cells were cultured in a specialized medium developed for human HSCs(Stemline II medium, Sigma, St. Louis, Mo.) supplemented with humanrecombinant IL-3, IL-6, and SCF. The human HSCs were then transducedwith retroviruses encoding MYC-ER or Bcl-2, along with a GFP reporter.The retroviruses were the same pMSCV variants used in Example 1.However, these retroviruses were modified to be packaged with anamphotropic envelope in order to enable transduction into human cells.The transduced human HSCs were either transplanted into sublethallyirradiated NOD/SCID/132M^(−/−) mice, or maintained in long-term culturesin vitro in the presence of the IL-3, IL-6, and SCF cytokine cocktailand 4-OHT. The two different approaches resulted in human ctlt-HSC celllines. A set of human ctlt-HSC cell lines generated completely in vitrohave been maintained in continuous culture for 14 months. The initialsurface phenotypes of three retrovirally transduced cell lines showed asignificant enrichment of the CD34⁺ fraction. The cells had over a10,000-fold enrichment of the CD34⁺ fraction over initial frequencies ofHSCs (FIGS. 3A-3C)

FACS analysis of was performed to determine the surface phenotype ofthree established human ctlt-HSC cell lines. As shown in FIG. 3, theexpanded and cryopreserved human ctlt-HSC cell lines retained a stablesurface phenotype that is represented. The transduced (i.e., GFP⁺) cellswere shown to express high levels of CD34 (FIGS. 3A-3C). In particular,the 3 different ctlt-HCS lines 5.23%, 5.09% and 4.03% GFPxCD34 doublepositive cells. These 3 ctlt-HSC lines were also 25.3%, 94.7, and 92.1cKitxGFP double positive (FIGS. 3D-3F). These same 3 ctlt-HSC lines alsoremained very low for lineage markers CD45, Flk-2, and CD150. Shown isthe flow cytometry characterization of 1 of the ctlt-HSC lines having12.3% CD45×GFP double positive cells (FIG. 3G), 0.77% Flk-2×GFP doublepositive cells (FIG. 3H), and 0.55% CD150×GFP double positive cells(FIG. 3I); as well as B220, CD19, and other lineage markers (thy1.2,Gr-1, Mac-1, and Ter-119). While there may be some heterogeneity interms of surface marker expression levels among the three establisheddifferent human ctlt-HSC cell lines, it is believed that theheterogeneity is the result of the previously reported inherentheterogeneity in the adult HSC compartment (McKenzie et al., Nat Immunol7, 1225-33, 2006).

In order to examine the pluripotency of the established human ctlt-HSCcell lines, a known xenotransplant model was used (Dick et al., StemCells 15 Suppl 1, 199-203, 1997). This model involves the irradiation ofNOD/SCID/β2M^(−/−) mice, and the transplantation of ctlt-HSC into theirradiated mice. Cohorts of NOD/SOD/β2M^(−/−) mice were sublethallyirradiated (300 Rads) and 10⁴ CD34⁺ human ctlt-HSC cells weretransplanted into the mice. The cells were maintained in culture for 10weeks prior to transplantation. The mice were bled at either 6 or 12weeks after transplantation. Lymphocytes present in the peripheral bloodof the humanized chimeric NOD/SOD/1321W mice were stained withantibodies specific to human leukocyte antigens. Specifically, thesamples were stained with antibodies to CD19, CD20 and CD3.

As shown in FIG. 4, human B cells (hIgM⁺ cells) and human CD45⁺ T cellswere detected in the peripheral blood of the chimeric NOD/SOD/1321Wmice, as compared to a control mouse that did not receive a stem celltransplant. In particular, peripheral blood of the control mouse had0.075% hIgM⁺/hCD45⁺ cells (FIG. 4A), peripheral blood of a firstchimeric mouse had 31% hIgM⁺/hCD45⁺ cells (FIG. 4B), peripheral blood ofa second chimeric mouse had 77.1% hIgM⁺/hCD45⁺ cells (FIG. 4C), andperipheral blood of a third chimeric mouse had 49.5% hIgM⁺/hCD45⁺ cells(FIG. 4B).

Example 5: In Vitro Generation of Mature RBCs from Human Ctlt-HSCs

The ability of human ctlt-HSCs to generate mature RBCs in vitro wasshown using the well-established surface markers of the erythroidlineage. Human ctlt-HSCs were incubated in liquid medium (Stemline IImedium), and treated with EPO and IL-3 for 12 days. As shown in FIGS. 5,H and E staining analysis 10 days after seeding the cultures with EPOand IL-3 showed a large number of enucleated cells in culture.Examination of enucleated cell populations by flow cytometry showed apopulation of cells expressing erythroid cell surface markersGlycophorin A, CD71, and CD41. Additionally, these cells lacked CD45expression or expression of other non-erythroid lineage markers.

In particular, FIG. 5A shows H and E staining of control mouseperipheral blood. FIG. 5B shows H and E staining of primary human fetalcord blood. FIGS. 5C, 5D, and 5E show H and E staining of threeconditionally transformed fetal cord blood cell lines that were treatedfor 12 days with IL-3 and EPO; and FIG. 5F shows a magnified view of thecells from FIG. 5E to show red blood cell morphology.

Example 6: In Vivo Functional Characterization of Ctlt-HSC-Derived HumanRBCs

The functionality of RBCs generated in vitro from ctlt-HSCs wasdetermined by testing for their ability to rescue mice from lethalanemias. As human RBCs were being used, the mice chosen for theseexperiments were immunocompromised mice. Two different strains ofimmunocompromised mice were used for these studies. These two micestrains are generally used for such studies (Hogan et al., Biol BloodMarrow Transplant 3, 236-46, 1997). The two strains are NOD/SCID miceand Rag-1^(−/−)/γc^(−/−) mice. The operating principle for the in vivofunctional studies was to induce some form of anemia that wouldotherwise be lethal in the mice, unless they are provided with afunctional RBC population.

One protocol that was used was adapted from Hiroyama (see, Hiroyama,PloS One 2, e1544, 2008). This protocol uses phenylhydrazine to induceanemia by hemolysis in vivo. The treated mice are then either given RBCsthe following day, or no further treatment. A second injection of thedrug follows 4 days later. If no functional RBCs are provided after thefirst round of chemically-induced hemolysis, the animals die soon afterthe second round and lethal challenge. If functional RBCs are provided,the mice are rescued from the lethal anemic challenge.

Ten days after initial seeding, 10⁷ human RBCs were derived in vitrofrom populations of human ctlt-HSCs using methods described herein. Acohort of NOD/SCID mice were obtained and treated with 80 mg/kg ofphenylhydrazine on day 0. On day 1, the 10⁷ human RBCs were transferredby tail vein injection into half the cohort of treated mice. The secondphenylhydrazine challenge was performed on day 6. The mice were thenobserved for survival for 9 days following the second phenylhydrazinechallenge.

As shown in FIG. 6, the ctlt-HSC-derived human RBCs were able to rescuethe mice from a chemically induced lethal anemia. In particular, onlyapproximately 20% of the mice treated with phenylhydrazine (Phenyl) butnot with human ctlt-HSCs-derived RBCs (No AT) survived, while 100% ofthe mice treated with both phenylhydrazine (Phenyl) and humanctlt-HSCs-derived RBCs (AT) survived (FIG. 6). This was comparable towild type mice that were not treated with phenylhydrazine (WT).

Another method that was used to test the in vivo functionality of thectlt-HSC-derived human RBCs included a protocol that is normally used toassess RBC homeostasis. In this instance, mice were bled extensively(5000, or about 17% total blood volume), and then left in the vivariumfor observation. However, it was discovered that normal mice recoveredfrom this injury without further intervention. It was thus reasoned thatin order to delay the recovery of the RBC compartment from hemorrhagicshock, the erythroid progenitors would need to incapacitated.

Accordingly, in order to affect the rate of RBC recovery,immunocompromised mice were sublethally irradiated with 450 Rads priorto 400 μl tail bleedings. In this instance, the mice died within 48hours unless a transfusion of 1×10⁷ erythroid cells was provided viatail vein injection. This form of lethal anemia was induced in a cohortof Rag-1^(−/−)/γc^(−/−) mice, with half the cohort receiving 1×10⁷erythroid cells derived in vitro from populations of human ctlt-HSCsusing methods described herein. The mice were monitored for survival for9 days following induction of the acute anemia and erythroid celltransplantation. As shown in FIG. 7, in vitro ctlt-HSC-derived humanRBCs were able to rescue the Rag-1^(−/−)/γc^(−/−) mice from the combinedinjury-induced lethal anemia. In particular, only approximately 10% ofthe Rag-1^(−/−)/γc^(−/−) mice survived the combined injury-inducedlethal anemia (Trauma No AT), while 100% of the mice rescued with humanctlt-HSCs-derived RBCs (Trauma AT) survived (FIG. 7). This wascomparable to wild type mice that were not subjected to the combinedinjury-induced lethal anemia (WT).

Example 7: Direct Protein Transduction of TAT-MYC and TAT-Bcl-2 FusionProteins into HSCs

One of the risks associated with the approach of utilizing MYC-ER and/orBcl-2-ER to generate ctlt-HSC cell lines is the random integration ofviral sequences into the genome of the host cells. This is a concern, asthe persistence of any nucleated ctlt-HSCs in the RBC preparations fortransfusion may pose an unwanted risk for patients receiving thosecells. The experiments described in this example demonstrate analternative approach for generating ctlt-HSC cell lines withoutintroducing viral sequences into the genome of host cells.

To introduce proteins into cells without genetic manipulation (i.e.,viral transduction), this alternative approach relies on the ability ofthe HIV-1 TAT (TAT) protein to cross biological membranes and deliver aprotein cargo into cells (Schwarze et al., Trend Pharmacol Sci 21, 45-8,2000). A number of plasmids were generated that encode TAT fragmentsfused to either MYC or Bcl-2. The plasmids were then transformed intobacterial cells, and the cells were induced with IPTG during log-phasegrowth. The induced cells were collected 3 hours later and the proteinswere purified on a Nickel column. Fractions were then analyzed by aBradford assay for protein content, and run on an SDS-PAGE gel that wasstained with Commassie Blue (FIG. 8). As shown in FIG. 8A, fractionsE2-E11 contained TAT-MYC, with fraction E3-E5 containing the mostTAT-MYC. As shown in FIG. 8B, fractions E1-E6 contained TAT-Bcl-2, withfraction E2 containing the most TAT-Bcl-2.

The notion of using TAT-MYC and TAT-Bcl-2 to directly transduce murineLt-HSCs was tested in order to generate ptlt-HSC cell lines withoutretroviral gene transduction of MYC-ER and Bcl-2. 5FU-enriched HSCs werecollected from the bone marrow of C57/BL6 mice and incubated in mediumthat was supplemented with recombinant IL-3, IL-6, and SCF.Additionally, the cells were incubated with purified recombinant 5 ug/mlTAT-MYC and 10 ug/ml TAT-Bcl-2 proteins that were prepared under lowendotoxin conditions. The medium and TAT-Fusion proteins were replacedevery 48 hours and the cells were maintained in culture for 21 days. Analiquot of the ptlt-HSC cell line was then used to characterize thephenotype of the murine ptlt-HSC cell line by flow cytometry. The cellswere stained with antibodies against stem cell markers c-kit and sca-1,as well as lineage markers CD3, B220, and Ter119. As shown in FIG. 9,the c-kit⁺, sca-1⁺, lin⁻ cell population in these cultures waspreferentially expanded. This is a similar phenotype to what is seen inprimary murine Lt-HSCs (Cheshier et al., Proc. Natl Acad. Sci. USA 96,3120-3125, 1999). In particular, 77.5% of the cells were c-kit⁺ andsca-1⁺ (FIG. 9B), while 98.1% were CD3⁻ and B220⁻ (FIG. 9C) and 99.4% ofthe cells were Ter119⁻ (FIG. 9D).

In order to characterize the in vivo pluripotency of the TAT-MYC andTAT-Bcl-2 derived ptlt-HSC cell line, 10⁴ cells were transplanted intosublethally irradiated Rag-1^(−/−) mice. Four weeks post transplant,peripheral blood was collected from the recipient mice by venipuncture.The PBMCs were then assessed by flow cytometry for B cell markers B220and IgM, and T cell markers CD4, CD8, and TCRβ. The stained cells werecompared to unstained cells. As shown in FIG. 10, transplantation of themurine ptlt-HSC cell line into the sublethally irradiated Rag-1^(−/−)mice resulted in reconstitution of the lymphoid compartment. Inparticular, approximately 10.7% of PBMCs were B220⁺ and IgM⁺ as comparedto 0% in the control Rag-1^(−/−) mouse (FIG. 10B); approximately 13.5%of PBMCs were CD4⁺ and TCRβ⁺ as compared to approximately 0.09% in thecontrol Rag-1^(−/−) mouse (FIG. 10C); and approximately 6.8% of PBMCswere CD8⁺ and TCRβ⁺ as compared to approximately 0.011% in the controlRag-1^(−/−) mouse (FIG. 10D).

Example 8: Development of RBCs from Murine ptlt-HSCs Transduced withTAT-MYC and TAT-Bcl-2 Fusion Proteins

Mature, anucleated RBCs were also derived from murine ptlt-HSC celllines generated by transduction with TAT-MYC and TAT-Bcl-2 fusionproteins, similar to what was observed with human ctlt-HSCs (FIG. 5). Inthis experiment, murine ptlt-HSCs were cultured both in the presence andabsence of IL-3, EPO, and TAT-MYC. After 10 days in culture, celldifferentiation was assessed by H and E staining (FIG. 11). It was foundthat murine ptlt-HSCs treated with IL-3, EPO, and TAT-MYC resulted in alarge frequency of fully enucleated murine RBCs (FIGS. 11B and 11C),while control cells did not differentiate into RBCs (FIG. 11A). Inaddition, the murine RBCs were characterized by flow cytometry and foundto express increased levels of Glycophorin A and decreased levels ofCD71.

Example 9: Generation of Human ptlt-HSC-Derived Red Blood Cells

The following example describes the in vitro production andcharacterization of human mature, enucleated red blood cells from ahuman protein transduced long-term HSC (ptlt-HSC) cell line.Advantageously, human red blood cells can be reliably produced in 10days under culture conditions that do not require the use of geneticallymodified HSCs, animal serum, or animal feeder cells. Additionally, theproduced red blood cells are fully differentiated and mature human redblood cells that are enucleated, express Glycophrin A (GPA), anddecreased levels of CD71 and fetal hemoglobin. The CD71 marker is thetransferrin receptor, which is normally expressed at high levels inerythrocyte (i.e., red blood cell) progenitor cells, but isdown-regulated in mature erythrocytes. The GPA marker is commonlyexpressed at high levels in mature erythrocytes as a sign of membranematuration.

In Vitro Production and Expansion of ptlt-HSC Cell Line

Transgenic and protein-transduced conditionally immortalizedhematopoietic stem cells have been described previously (references). Inthis experiment, ptlt-HSC cell lines were produced using proteintransduction with TAT-MYC and TAT-Bcl-2 fusion proteins as describedpreviously. These fusion proteins contain a TAT peptide derived from theHIV-1 TAT (TAT) protein.

A unit of human cord blood was obtained from a local cord blood bank.The nucleated cell population from the cord blood was then isolated bydiluting the cells 1:1 with phosphate buffered saline (PBS). 20 ml ofdiluted cord blood cells are gently overlaid onto 20 ml Ficoll-PaquePlus (Amersham Biosciences). The cells were then spun at 900× gravityfor 60 minutes. After the spin, the huffy coat was removed with a glasspipette and washed twice with PBS. The cells were then resuspended andcultured in Iscove's Modified Dulbecco's Medium supplemented with 15%human plasma, 100 units per ml Penn/Strep, 20 ng/ml IL-3, 50 ng/ml IL-6,50 ng/ml Stem Cell Factor, 20 ng/ml GM-CSF, 20 ng/ml TPO, 20 ng/mlFlt3-L, 5 μg/ml TAT-MYC and 5 μg/ml TAT-Bcl2.

The initial cell population (Day 0) was 0.12% CD34⁺/CD38^(lo). After 3days in culture, the frequency of CD34⁺/CD38^(lo) cells had risen to1.24% (FIG. 12A). After 14 days in culture, 45.2% of the cell populationwas CD34⁺/CD38^(lo) (FIG. 12B). Moreover, there was a net increase inthe total number of cells after 14 days in culture. The HSCs formed acell line after being expanded in culture for 21 days in the presence ofTAT-MYC and TAT-Bcl-2. This cell line was designated as a proteintransduced long-term HSC (ptlt-HSC) cell line. This cell line was thenused as the source of cells for red blood cell (RBC) differentiation andcharacterization.

In Vitro Production of ptlt-HSC-Derived RBCs

The in vitro produced human ptlt-HSCs were then used to produce humanenucleated and mature RBCs. These ptlt-HSC-derived human RBCs were thencharacterized. FACS analysis was used to measure the expression levelsand patterns of human Glycophrin A (GPA), human CD71 (transferrinreceptor), and human fetal hemoglobin.

Red blood cell differentiation is induced by culturing the cordblood-derived ptlt-HSCs in DMEM medium supplemented with 3.2 ng/ml IL-3and 100 units/ml EPO, as well as 15% human Plasma and 100 units per mlPenn/Strep. The cells were then cultured for at least 9 days.

As shown in FIG. 13, upon transfer of the ptlt-HSC culture to RBCdifferentiation conditions (IL-3 and EPO), the cells began to expressGPA and CD71 at high levels after 4 days. Cells maintained in mediumcontaining TAT-MYC and TAT-Bcl2, but lacking IL-3 and EPO, did not showthese changes (FIG. 12). A sample of the culture was used for FACSanalysis of cell surface expression of Glycophrin A (GPA) and CD71(transferrin receptor). As shown in FIG. 13, ptlt-HSCs induced into theRBC differentiation program by culturing in the presence of IL-3 and EPOwere stained for GPA and CD71 expression, and compared to unstainedcells and ptlt-HSCs cultured in a neutral medium containing TAT-MYC andTAT-Bcl2, but lacking IL-3 and EPO. The results show that by day 4 ofculturing with IL-3 and EPO, approximately 42% of cells were CD71⁺/GPA⁺(FIG. 13C), as compared to the approximately 0.366% of CD71⁺/GPA⁺ cellsin the unstained control (FIG. 13A) and the approximately 0.222% ofCD71⁺/GPA⁺ cells that were cultured for 4 days in the neutral media(FIG. 13B).

Additionally, 9 days after transitioning the ptlt-HSCs intodifferentiation culture, the developing RBCs began to reduce theirexpression of the transferrin receptor (CD71), while retaining highlevels of GPA on their surface (FIG. 14). This shift in the expressionof CD71 was concurrent with an increase in the frequency of enucleatedhuman RBCs in the culture (FIGS. 14 and 15). The cells were also stainedfor fetal hemoglobin.

FIG. 14 shows that as the developing RBCs transition from GPA/CD71double positive to GPA⁺/CD71^(lo), they also switch from expressing highlevels of human fetal hemoglobin to low levels of fetal hemoglobin.Without wishing to be bound by theory, it is believed that theGPA⁺/CD71^(lo) cells having low levels of fetal hemoglobin havecorrespondingly high levels of adult hemoglobin.

FACS analysis of cells from the RBC differentiation cultures wasperformed at three time points after induction (day 7, day 12 and day22). The FACS analysis measured cell surface expression of GPA and CD71.The cells were also monitored for expression of fetal and adulthemoglobin. Primary human RBC obtained from peripheral blood were usedas a positive control (FIG. 14; Control top row). The results show thaton day 7, there were many GPA/CD71 double positive cells (FIG. 14; 1stpanel top). The results also show that by day 12, the cells had mostlyshifted to GPA⁻/CD71^(lo) (FIG. 14; 2nd panel top). The cells from day12 also expressed high levels of fetal hemoglobin (FIG. 14; 2nd panelbottom). By day 22 in differentiation media the cells remainedGPA⁺/CD71^(lo) but had downregulated expression levels of fetalhemoglobin and increase expression of adult hemoglobin (FIG. 14; 3rdpanel top and bottom). These dynamic changes of maturation markerexpression were consistent with changes observed in bone marrow duringnormal RBC differentiation in humans and mice. In particular, after 7days of culturing the ptlt-HSC cells in induction culture (IL-3 andEPO), approximately 60.9% of cells were GPA⁻/CD71⁺, approximately 1% ofcells were GPA⁺/CD7F, and 71.4% of cells expressed fetal hemoglobin(FIG. 14B). After 22 days of culturing the ptlt-HSC cells in inductionculture (IL-3 and EPO), the GPA⁺/CD71⁺ had transitioned to GPA⁺/CD71⁻.Similar to the control blood, 76% of the GPA positive cells expressedadult hemoglobin and only 20.3% of cells expressed fetal hemoglobin(FIG. 14; 2nd and 3rd panel bottom row).

Additionally, histological analysis of anucleation in culture wasperformed by staining day 3 and day 7 RBC differentiation culturesamples with H and E (FIG. 15). Cells were laid on a slide using acytospin apparatus. The slides were then stained with H and E, andphotographs were obtained using an inverted microscope and lightphotography. The appearance of small, enucleated cells is observed asearly as day 3 (FIG. 15A), with increased numbers seen by day 7 (FIG.15B).

Example 10: Scale-Up Production of ptlt-HSC-Derived Red Blood Cells

The following example describes scaling up the in vitro production ofhuman ptlt-HSC-derived RBCs to clinically relevant levels of production.

Production of ptlt-HSC-Derived RBCs in Gas Permeable Bag

A gas-permeable bag was also used to scale up production of humanptlt-HSC-derived RBCs in vitro (FIG. 16).

A gas-permeable bag having a Teflon-based coating on the cell contactside was optimized for the in vitro production of the ptlt-HSC-derivedRBCs (FIG. 16A). The culture was started with human ptlt-HSCs that wereincubated in neutral conditions, with TAT-MYC and TAT-Bcl-2, for 5 daysin a gas-permeable bag (Origene). The culture was then switched intomedium containing IL-3 and EPO in order to induce RBC differentiation.The photo shown in FIG. 16A was taken after 4 days of incubation in theRBC differentiation medium. FIG. 16B shows RBC maturation andanucleation in the gas-permeable culture bags. Cells were sampled fromthe bag shown in FIG. 16A, and fixed to a glass slide with a cytospinapparatus. The slide was then stained with H and E, and a photographobtained using an inverted microscope and light photography.

Serial Passage of In Vitro ptlt-HSC Differentiation Culture

This example describes the length of time ptlt-HSCs cultured in RBCdifferentiation medium can continue to produce RBCs in vitro. This is animportant factor in the development of a process for producing aclinically relevant amount of RBCs for human transfusion (10¹¹cells/unit).

The first set of experiments involves the serial passage of ptlt-HSCsunder erythroid differentiation conditions (i.e., cultured with IL-3 andEPO) in vitro. Ctlt-HSCs are plated at densities of 3×10⁶, 10⁶, 3×10⁵,and 10⁵ in a 6 well plate. The cells are cultured in Iscove's ModifiedDulbecco's Medium supplemented with 10% human plasma, 100 units per mlPenn/Strep, 20 ng/ml IL-3, 50 ng/ml IL-6, 50 ng/ml Stem Cell Factor, 20ng/ml GM-CSF, 20 ng/ml TPO, and 20 ng/ml Flt3-L. The starting ptlt-HSCpopulation is then stained for human HSC and erythroid markers (GPA,CD71 and fetal hemoglobin). The cultures are monitored visually and byFACS analysis for the development of mature RBCs. An aliquot of thecells is removed 10 days after the initial cultures are set up, and spunthrough a Ficoll gradient in order to separate the nucleated live cellsfrom the RBCs. Both fractions of cells are analyzed by FACS forexpression of cell surface markers of HSC and erythroid lineages. Asimilar approach is also used for analysis of the cultures at the end ofeach 10-day cycle.

The nucleated cells present in the interphase of the Ficoll gradient arewashed in fresh PBS and medium, and plated again under the sameconditions. It was shown that at least one ctlt-HSC cell line was ableto generate mature RBCs after three serial passages, starting with asingle concentration of ctlt-HSCs. In some embodiments, ctlt-HSC linesare expanded and then exposed to red blood cell differentiation media inthe presence of PTD-Myc. After red blood cells are separated inapproximately 9 to 28 days, the remaining non-red blood cells are againplaced in differentiation media, and red blood cells are separated inapproximately 9 to 28 days. This process can be repeated at least 3times, and is expected to continue indefinitely

The results suggest that ptlt-HSC-derived erythroid progenitors cancontinue to produce RBCs for 2-4 passages.

Large-Scale Production of ptlt-HSC-Derived RBCs in a Culture BioreactorSystem

This example describes testing four different systems for thelarge-scale in vitro production of mature, enucleated human RBCs fromptlt-HSCs.

Two systems were initially tested (a flexible plastic container designedfor cell culture and a gas-permeable bag). The results with thesesystems showed that large-scale human RBC production can be optimized bymaking design changes in the tissue culture vessels and alterations tothe culture conditions.

In order to consider more efficient approaches for the generation oflarge numbers of RBCs, the one step protocol described above forproducing RBCs in vitro from human ptlt-HSCs is adapted to spinner flaskbioreactors (e.g., Ambr micro bioreactor from TAP Biosystems, andIntegrity PadReactor from ATMI), gas-permeable bags (Origene system),gas-permeable tissue culture flasks (Wilson Wolf G-rex Oxygen permeableflasks), or flexible plastic containers designed for cell culture (GEWave system). These experiments determine the feasibility of large-scaleproduction of human RBCs in vitro from ptlt-HSCs.

The basic protocol used to generate RBCs in vitro from human ptlt-HSCsbegins with obtaining cord blood-derived HSCs. The nucleated cellpopulation from umbilical cord blood is isolated by diluting the cells1:1 with phosphate buffered saline (PBS). 20 ml of diluted cord bloodcells are gently overlaid onto 20 ml Ficoll-Paque Plus (AmershamBiosciences). The cells are then spun at 900× gravity for 60 minutes.After the spin, the huffy coat is removed with a glass pipette andwashed twice with PBS. The cells are then resuspended in Iscove'sModified Dulbecco's Medium supplemented with 10% human albumin, 100units per ml Penn/Strep, 20 ng/ml IL-3, 50 ng/ml IL-6, 50 ng/ml StemCell Factor, 20 ng/ml GM-CSF, 20 ng/ml TPO, 20 ng/ml Flt3-L, 5 μg/mlTAT-MYC and 5 μg/ml TAT-Bcl2. The cells are maintained under theseculture conditions for 12-30 days, depending on the degree of enrichmentfor the CD34+/CD38− population desired, as well as the total cell numberrequired. Red blood cell differentiation is induced when the medium ischanged to DMEM supplemented with 15% human albumin, 100 units per mlPenn/Strep, 20 ng/ml IL-3, and 3.2 units/ml EPO. Cells are cultured foran additional 11 days. Cells are then monitored for RBC differentiationby FACS using antibodies against GPA, CD71, and fetal hemoglobin.Histology staining for H and E is also performed.

The in vitro RBC production protocol is adapted for large-scaleproduction in a spinner flask-based bioreactor that was previously usedto grow large numbers of human hematopoietic cells derived from cordblood units. This system involves the use of two different spinner flaskbioreactor systems. First, the Ambr micro bioreactor system (TAPBiosystems) is used. This is an apparatus that carries 10-15 ml culturesunder spinner flasks conditions that mimic the characteristics ofclassical bioreactors, on a small scale. This instrument uses disposablemicro reactor chambers and is controlled in an automated manner. One ofthe key advantages is that it allows for the simultaneous culture of 24different conditions (Genetic Engineering and Biotechnology News, Nov.1, 2010, Vol. 30, N. 19). This approach enables the quick optimizationand adaptation of the in vivo conditions for RBC development to abioreactor based format. Once the conditions are optimized, the processis transitioned to a large scale system (Integrity PadReactor, ATMI).The PadReactor system is a single use bioreactor that is composed of adrive unit, which allows the user to simultaneously grow cells in bagsof different volumes, a mobile tank, that supports the bag in which thecells grow and is able to move into modular manufacturing spaces, aswell as the bioreactor vessel, which is a single use cell bag thatcontains a paddle and allows for non-invasive mixing as the paddlerotates inside the bag. This system provides improved mixing with reduceshear forces, is amenable to suspension cells, and can grow the cells ina low volume.

Gas Permeable Bags

As described above, a basic gas-permeable bag (Origene) was used toproduce RBCs in vitro. These bags have an interior Teflon coating thatis also able to exchange gas from the entire surface area of the vessel.However, the system can be optimized to continuously provide mediumchanges once the cells reach a critical density. Accordingly, a newsystem can be designed that enables continuous flow of medium in atwo-chamber gas-permeable bag system. In addition to minimizing cellloss, the system is also able to provide for a continuous flow systemwhere medium and waste are perfused through the outer chamber. Thissystem also allows the switching of ptlt-HSC cultures from HSC growthconditions to RBC differentiation conditions.

The ptlt-HSC are tested to determine whether the cells are able topropagate in the dual bag system. The bag is seeded with 10⁷ humanptlt-HSCs and maintained in Iscove's Modified Dulbecco's Mediumsupplemented with 10% human albumin, 100 units per ml Penn/Strep, 20ng/ml IL-3, 50 ng/ml IL-6, 50 ng/ml Stem Cell Factor, 20 ng/ml GM-CSF,20 ng/ml TPO, and 20 ng/ml Flt3-L along with TAT-MYC and TAT-Bcl-2.These are neutral conditions that allow the ptlt-HSC cells to propagateand retain their pluripotency. The initiating ptlt-HSCs are then stainedwith antibodies for human stem cell surface markers 10 days later andthe magnitude of the expansion of the ptlt-HSCs is determined underthese conditions.

Once the dual bag is tested in the context of supporting the expansionof ptlt-HSC under neutral conditions, a dual bag system will be seededwith ptlt-HSCs for RBC production. The pore size used is in the order ofa SkDa cutoff, to prevent the RBCs from being flushed during the cyclingof the medium. The dual bag system will be seeded with 10⁷ ctlt-HSCs andthe cells will be cultured with Iscove's Modified Dulbecco's Mediumsupplemented with 10% human albumin, 100 units per ml Penn/Strep, 20ng/ml IL-3, 50 ng/ml IL-6, 50 ng/ml Stem Cell Factor, 20 ng/ml GM-CSF,20 ng/ml TPO, and 20 ng/ml Flt3-L along with TAT-MYC and TAT-Bcl-2.After 3 days in culture, the medium is switched to Iscove's ModifiedDulbecco's Medium supplemented with 10% human albumin, 100 units per mlPenn/Strep, 20 ng/ml IL-3, and 50 ng/ml EPO. Samples are collected fromthe bioreactor every 3 days. This time frame is a result of the need toreplenish some of the medium with cytokine containing medium. Thecollected cells are then analyzed by counting as well as by FACS andmicroscopy as described in the Examples above. The total output of RBCsis determined over the period of time (10-12 days) that the dual bagsystem is reported to support cell expansion. In addition, the fate ofthe ptlt-HSCs that were used to inoculate the cartridge on day 1 is alsodetermined. Ideally, the ptlt-HSCs remain active for 2-4 rounds ofculture in the dual bag system such that the RBCs can be harvested andthe remaining ptlt-HSCs can be re-seeded in fresh medium in order togenerate additional RBCs. Continuous centrifugation methods currentlyused for RBC separation from peripheral blood obtained from patients isused for the collection of the RBCs generated in the dual bag system.

Flexible Plastic Bag Containers

As described above, a flexible plastic bag container designed for cellculture (GE Wave) was also used to produce RBCs in vitro. However, thesystem can be optimized to reduce the premature and uncontrolleddifferentiation of the starting ptlt-HSC population.

A large number of ptlt-HSCs obtained from static cultures in standardvented tissue culture flasks is transferred into RBC differentiationmedium when placed into the flexible plastic bag container. The rates ofanucleation and kinetics of RBC maturation are examined by using FACSanalysis to measure expression levels of CD71, GPA, and fetalhemoglobin. Histological analysis is also used to verify the state ofmaturation. The interior of the flexible plastic container is thencoated with a small number of ptlt-HSCs that are expanded and switchedto RBC differentiation medium in the same vessel.

Gas Permeable Tissue Culture Flask

In addition to experimenting with two different bag-based bioreactorsystems, and a spinner flask apparatus, a gas-permeable tissue cultureflask is tested for scaling up production of ptlt-HSCs and RBCs.

A gas-permeable flask (Wilson Wolf) is tested for both scaled productionof a small number of ptlt-HSCs into a clinically relevant number ofcells, and regulated differentiation of ptlt-HSCs into RBCs. The entireprocedure is performed in a single flask. The flask provides cells withfar better access to oxygen and nutrients than existing devices.Furthermore, the flask is much easier to use. The flask functions in astandard incubator and uses standard laboratory equipment. The bottom ofthe flask is made of a unique di-methyl silicone gas-permeable membranethat provides better oxygen permeability than any existing gas-permeabledevice (Lapteva and ad Ver, Stem Cells Int, Epub 2011, September 11).Cells gravitate to this gas-permeable membrane, where they are submergedunder far greater medium depth than existing devices can allow. Underthese conditions, the cells receive oxygen and nutrients on demandwithout being subjected to frequent feeding or any disturbances frommedium mixing equipment.

Example 11: In Vitro and In Vivo Characterization of Human ptlt-HSC CellLines

The following example describes the characterization of the surfacephenotypes, and in vitro and in vivo pluripotency of the human ptlt-HSCcell lines produced in Example 9. The experiments described in thissection determine the lineage potential in vitro using standardmethycellulose differentiation assays, and in vivo using xenotransplantmouse models.

A minimum of 4 human ptlt-HSC cell lines are used for the studiesdiscussed in this example. The human ptlt-HSC cell lines selected arechosen based on the following four criteria. First, the cell lines havea surface phenotype that resembles primary human Lt-HSCs (CD34⁺, CD133⁺,CD48⁺, CD150⁺, lin⁻). Second, the human ptlt-HSCs propagate vigorouslyin culture under the conditions described in Example 9, and retain adependency on exogenously added TAT-MYC for proliferation in vitro.Third, the human ptlt-HSCs are able to recover from cryopreservation ina prompt manner while retaining their surface phenotype and growthcharacteristics. Fourth, one cell line is selected from each of thefollowing genotypes: A rh−, B rh−, AB rh−, O rh− in order to generate apanel of human ptlt-HSCs that give rise to the major forms of RBCs thatare used clinically.

Ten, 10², or 10³ human ptlt-HSC cell lines are seeded in methycellulosedifferentiation medium as previously described (Dick et al., Stem Cells15 Suppl 1, 199-203, 1997). Medium that is supplemented with cytokinesintended to push HSC differentiation toward the myeloid lineages (Dicket al., Stem Cells 15 Suppl 1, 199-203, 1997), myeloid-erythroidlineages, or pre-B-cell lineage (Cheshier et al., Proc. Natl Acad. Sci.USA 96, 3120-3125, 1999; and Hogan et al., Biol Blood Marrow Transplant3, 236-46, 1997) is specifically used. The plates are then evaluated forcolony formation in terms of number of colonies, morphology, andkinetics of colony development as a measurement of precursor frequency.Ptlt-HSC cell lines that are pluripotent, as defined by their ability togive rise to specific colonies in each of the different conditionstested, are then identified.

Once it has been demonstrated that the ptlt-HSC cell lines in questionare competent in their ability to give rise to multiple hematopoieticlineages in vitro, their pluripotency is examined in vivo. Theseptlt-HSC cells are then used for transplantation into cohorts of 10sublethally irradiated NOD/SCID mice, as previously done with primaryhuman Lt-HSCs (Dick et al., Stem Cells 15 Suppl 1, 199-203, 1997; andHogan et al., Biol Blood Marrow Transplant 3, 236-46, 1997). Theirradiated mice are given transplants of 10⁴ ptlt-HSCs that have beenmaintained in the culture conditions described in Example 9. The miceare then bled by venipuncture in order to collect peripheral bloodsamples for analysis. The red blood cells are lysed, and the PBMCs arestained for human CD3 and CD19. Once human lymphoid cells are detectedin the peripheral blood, the mice are euthanized by CO₂ asphyxia andcervical dislocation. Lymph nodes, spleen, thymus, and bone marrow arethen harvested from the mice. Single cell suspensions are generated fromthe organs and the cells are stained with antibodies specific for humanCD3, CD19, CD4, CD8, Mac-1, Gr-1, and Ter-119. The detection of multiplelineages of human hematopoietic cells confirms the pluripotency of thehuman ptlt-HSC cell lines. Two controls are included. For the negativecontrol, non-manipulated mice, or mice that are sublethally irradiatedand not given a transplant of human ptlt-HSC cells are used. As apositive control, a cohort of mice that are sublethally irradiated andtransplanted with the human ctlt-HSC cell line generated in Example 4are used.

Example 12: Analysis of Hemoglobin Types Expressed in ctlt-HSC-DerivedHuman RBCs

The following example describes the differentiation state (e.g., theextent of differentiation into the erythroid lineage, and thedevelopmental state) of the human red blood cells (RBCs) produced inExample 9, by determining the specific kind of hemoglobin (fetal, adult,etc.) of the RBCs.

The nature of the hemoglobins expressed in the human RBCs generated inExample 11 using methods described herein is determined by two parallelapproaches. First, mRNA is obtained from the RBC precursors present inthe cultures used to generate human RBCs. In addition, primary humanRBCs obtained from a healthy, anonymous volunteer are also used as apositive control. Human ctlt-HSCs are incubated in Stemline II mediumsupplemented with IL-3 and EPO, as described in Example 7. Fractions ofcells are collected every 48 hours for 10 days. A fraction of the cellsare used for flow cytometric analysis for cell surface phenotype anderythroid differentiation markers. The cells are stained for human CD71and GPA. The remainder of the cells in the sample is used to obtain mRNAin order to generate cDNA. The resulting cDNAs are used as templates forsemiquantitative RT-PCR (Q-PCR) for three globin genes (hemoglobin α, β,and γ), and two housekeeping genes (β-actin and GAPDH). Sets of primersare used to amplify hemoglobin transcripts that are normally expressedin fetal RBCs. mRNA from the positive control is also isolated. It isexpected that the human RBCs and their progenitor cells generated invitro from ctlt-HSCs express adult globin genes. The mRNA results areconfirmed by using monoclonal antibodies and FACS analysis.

The presence of hemoglobin proteins in the ctlt-HSC-derived humans RBCsare confirmed by perfusion chromatography and HPLC, as previouslydescribed (Honig et al., J Biol Chem 265, 126-32, 1990). The RBCsobtained from in vitro cultures described above are collected and washed3 times in 0.9% NaCl, then suspended in 9 volumes of water, lysed withsaponin and clarified by centrifugation at 600×g. Globin mass spectra isobtained using a MALDI-TOF (matrix assisted laser desorption/ionizationtime-of-flight) mass spectrometer (Bruker Omniflex), as describedpreviously (Honig et al., Am. J. Hematol 34, 199-203, 1990). ZipTips ispurchased from Millipore and packed with C18 and C4 resins to preparethe solutions for MS analysis of peptide and protein, respectively.Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA) are used asthe matrix for peptide and protein, respectively. Aliquots (1.3 ml) ofthe matrix solution (3-10 mg CHCA or SA in 1 ml aqueous solution of 50%acetonitrile containing 0.1% TFA) are used to elute the peptide/proteinfrom ZipTips and spotted onto MALDI-TOF target. A LC/MS/MS system(Agilent series 1200 HPLC modules, Agilent HPLC Chip interface, Agilent6510 Quadrupole Time-of-Flight mass spectrometer) equipped with a 337 nmpulsed nitrogen laser is used to analyze the samples. External masscalibration is performed using the peaks of a mixture of pigeoncyctochrome c at m/z 12362, apomyoglobin at m/z 16952 and adolase(rabbit muscle) at m/z 39212.

The mRNA levels for globin genes that are normally expressed in fetalRBCs are also examined to compare against the globin genes expressed inthe ctlt-HSC-derived humans RBCs.

Example 13: Analysis of Oxygen-Binding Characteristics ofctlt-HSC-Derived Human RBCs

The following example describes the functional nature of the hemoglobinproteins expressed in the human red blood cells (RBCs) produced inExample 9, by measuring the Oxygen equilibrium curves in the RBCs.

Oxygen equilibrium curves as are measured as previously described (Honiget al., Am. J. Hematol 34, 199-203, 1990; Maurer et al., Nature 227,388-90, 1970; and Lee et al., Rapid Commun Mass Spectrom 19, 2629-35,2005). The method that is used is a continuous method using adouble-wavelength spectrophotometer (Hemox analyzed, TCS). The RBCs aresuspended in 50 mM bis-Tris buffer containing 140 mM NaCl at 37° C. andpH 7.4. The binding properties of hemoglobin are studied by flashphotolysis of solutions in 1-mm optical cuvettes. Briefly, the kineticsof the rebinding of CO to intracellular hemoglobin tetramers is analyzedat 436 nm after photolysis with a 10-ns pulse at 532 nm, as previouslydescribed (Honig et al., Am. J. Hematol 34, 199-203, 1990; Maurer etal., Nature 227, 388-90, 1970; and Lee et al., Rapid Commun MassSpectrom 19, 2629-35, 2005).

Oxygen binding characteristics of the ctlt-HSC-derived human RBCs isalso analyzed.

Example 14: Analysis of Cell Shape and Flexibility of ctlt-HSC-DerivedHuman RBCs

The following example describes the determination of whether the humanred blood cells (RBCs) produced in Example 9 can elongate and functionin the context of the microvasculature in vivo by measuring theflexibility of the RBCs.

The deformability of the ctlt-HSC-derived human RBCs and primary RBCsobtained from peripheral blood is examined as previously described (Kaulet al., Am J Physiol Heart Circ Physiol 295, 2008). Briefly, RBCspreparations are obtained from the in vitro ctlt-HSC cultures describedin Example 9, and from peripheral blood from a healthy adult are passedthrough a deleukocyting filter (Leucolab LCG2, Macopharma). Theenucleated cells are then examined by ekacytometry. The RBCs aresuspended in 4% polyvinylpyrrolidine solution and then exposed to anincreasing osmotic gradient in the ektocytometer (Technicon, Bayer)(from 60 to 450 mosM). The change in the laser diffraction of the RBCsin this setting is recorded. The photometric measurement produces asignal termed the deformability index (DI). Analysis of the DI curvesprovides a measure of dynamic deformability of the cell membrane as afunction of osmolarity at a constant applied shear stress of 170dynes/cm2. The DI max is related to the mean surface area of the cells.

Example 15: Analysis of Lifespan of ctlt-HSC-Derived Human RBCs

The following example describes the average lesion-free lifespan of thehuman red blood cells (RBCs) produced in Example 9 in order to ascertainif they are equivalent to primary human RBCs. The average lifespan of aprimary human RBC is estimated at 120 days. The shelf life of RBCconcentrates for clinical use is generally 28 days, due to the variationof ages of the RBCs collected from peripheral blood. It is believed thatthe ability to synchronize production of RBCs in vitro enables asignificant increase in the shelf life of RBC concentrates for clinicaluse.

In some embodiments, red blood cells are produced using the methodsdescribed herein over 7 to 30 days. In some embodiments, red blood cellsproduced using the methods described herein are collected on or aboutthe same day, for example, on or about Day 9, Day 10, Day 11, Day 12,Day 13, Day 14, Day 15, Day 16, Day 17, Day 18, Day 19, Day 20, Day 21,Day 22, Day 23, Day 24, Day 25, Day 26, Day 27, or Day 28. The red bloodcells are then assessed for viability over time using known methods,including those described herein.

In some embodiments, red blood cells would be maintained in red bloodcell storage media. In some embodiments, the red cell storage mediafurther includes Bcl-2, optionally PTD-Bcl-2. Bcl-2 (optionallyPTD-Bcl-2 may be provided as an initial bolus, may be providedcontinually, or provided at intervals (e.g. every 24 hours, 48 hours, 72hours, 96 hours, etc.). Concentrations of Bcl-2 provided may include 0.5ug/ml to 100 ug/ml or more. In some embodiments, 1 ug/ml, 5 ug/ml, 10ug/ml, 25 ug/ml, or 50 ug/ml of Bcl-2 (optionally TAT-Bcl-2) may beprovided to the storage media. The red blood cells are then assessed forviability over time using known methods, including those describedherein.

In order to determine the average lesion-free lifespan ofctlt-HSC-derived human RBCs, three criteria are used that can bequantified and measured by flow cytometry. First, the number of viableRBCs present in a culture is counted over a period of time, in order tomeasure the attrition rate from a known starting number. Second, thelevels of CD47 on the surface of the ctlt-HSC-derived human RBCs aremeasured. Third, the levels of phosphatidylserine (PS) exposed on thesurface of the ctlt-HSC-derived human RBCs are measured. All threecriteria are measured using a flow cytometric approach. The forward andside scatter characteristics are used in combination with a vital-dye(LDS-751) in order to ascertain the number of viable, enucleated RBCspresent in a culture at a particular point in time. The levels of CD47present on the surface of RBCs are measured using a monoclonal antibodythat is conjugated to the fluorochrome APC. The levels of PS exposed onthe surface of the RBCs are measured with Annexin V-FITC, as previouslydescribed (Holovati et al., Transfusion 48, 1658-68, 2008).

Cultures of ctlt-HSCs are set up in the presence of IL-3 and EPO, asdescribed in Example 9. The RBCs appear first on days 8-10 of theculture. RBCs from the ctlt-HSCs cultures are collected and passedthrough a deleukocyting filter (Leucolab LCG2, Macopharma). Primaryhuman RBCs are obtained from a healthy anonymous donor. Both sets ofcells are set up in culture in Stemline II medium, and the cultures aremaintained at 37° C. Aliquots of cells are also stored at 4° C. in acitrate buffer that is normally used to store RBC concentrates(Lagerberg et al., Transfusion 47, 2242-9, 2007). Each set of conditionsare established with 10¹⁰ cells.

In order to determine the lesion-free survival of the RBCs from eithersource, an aliquot is removed every 4 days and the cells are stainedwith the vital dye (LDS-751), anti-human CD47-APC, and Annexin V FITC.The cells are then analyzed using a BD FACSCalibur Flow cytometer.Aliquots are continuously analyzed until there are no more viable cellsleft in the specific condition, or for 120 days, whichever comes first.The RBC cultures maintained in Stemline II medium at 37° C. have theirmedium replenished every 7 days.

Example 16: Comparison of Methods for RBC Differentiation from 5-FUTreated Bone Marrow

The following example describes the production of red blood cells frombone marrow of mice.

C57BL/6 (Jackson labs #003548) mice are intravenously injected with 5 mg5-fluorouracil (Genera Medix Cat #NDC 10139-063-11) in 200 ul Dulbecco'sPBS. Five days later, bone marrow cells are harvested from the tibia andfemur of the C57BL/6 mice.

Pellet harvested bone marrow cells, and resuspend in 5 ml sterile TACbuffer (135 mM NH₄CL, 17 mM Tris Ph 7.65) to lyse the red blood cells.Allow cells to sit in TAC buffer for 1-2 min and then spin cells down at1200 RPM for 5 min. Wash the cells with 25 ml of D10 media. Resuspendcell pellet in 10 ml of BM Medium (500 ml bottle DMEM containing 90 mlheat inactivated FBS, 6 ml Penn/Strep (Gibco Cat #15140), 6 ml MEM NEAA(Gibco Cat #11140), 6 ml L-glutamine (Gibco Cat #25030), 6 ml Hepes(Gibco Cat #15630), and 60 ml of cytokine cocktail (IL3, IL6, and SCF).

These resuspended cells are counted and seeded in wells of a 24 wellcluster dish at a density of 1×10⁶ cells per well in 1 ml of medium.Note: if 5FU treatment works each mouse should yield 1-1.2×10{circumflexover ( )}6 BM cells compared to 10×10{circumflex over ( )}6 in anuntreated mouse.

Each well containing 1 ml of media is treated with 5 units of TAT-MYCand 5 units of TAT-Bcl2 diluted in 20 ul human serum albumin (GrifolsNDC 68516-5216-2). Media is changed every 2 days to refresh cytokinesand TAT-fusion proteins. Sca-1×cKit population will begin to dominatethe culture beginning around days 14-17.

Seed 2×10⁵ of the Sca-1×cKit cells per well of a 6 well plate. Replacethe BM media with RBC differentiation media #1 (IMDM supplemented to 15%heat inactivated FBS, 10% IL3 containing media, and 100 units per mlEPO, 100 mM dexamethasone and 25 ug/ml Holo-Tranferrin). To separatetest wells, add the following test fusion proteins: 5 units per/ml ofTAT-MYC, 5 units per/ml of TAT-Bcl-2, or both 5 units per/ml of TAT-MYCand 5 units per/ml of TAT-Bcl-2.

The RBC differentiation media #1 and fusion proteins are refreshed every2 days for the first 6 days. RBC media #1 is then replaced with RBCmedia #2 (IMDM supplemented to 15% heat inactivated FBS, 10% IL3containing media, and 100 units per ml EPO and 25 ug/mlHolo-Tranferrin). Continue to refresh RBC media #2 and fusion proteinsevery 2 days until RBCs appear, about 9-12 days.

As shown in FIG. 17, the addition of TAT-Myc (FIG. 17C) during red bloodcell differentiation leads to increased numbers and percent of red bloodcell production compared with untreated controls (FIG. 17A),differentiation in IL3 and EPO alone (FIG. 17B), or IL3 and EPO with theaddition of TAT-Bcl-2 (FIG. 17D) or the combination of TAT-Bcl-2 andTAT-Myc (FIG. 17E).

Example 17: Payloading Murine Red Blood Cells

5FU enriched bone marrow derived HSCs are collected as describedpreviously. The cells are collected 5 days after 5 FU treatment of mice,placed in culture with IL-3, IL-6, SCF and TAT-MYC and TAT-Bcl-2. Thecells are retrovirally transduced for the first 2 days of culture withone or more of pMSCV-hCD122-IRES-GFP or pMSCV-IRES-GFP. Followingtransduction, the cells are expanded in media containing IL-3, IL-6,SCF, TAT-MYC and TAT-Bcl-2 for another 14 days.

The cells are characterized by FACS for surface levels of c-kit, sca-1,lineage markers (B220, CD3, ter-119, Mac-1, Gr-1), as well as GFPexpression and surface hCD122. The expanded murine LSK population thatalso expresses GFP and hCD122 will then be switched to the RBCdifferentiation conditions using media containing IL-3, EPO and lowconcentrations of TAT-MYC as described previously. 14-28 days later, thecultures will be continuously monitored and characterized for expressionof CD71, GPA, adult and fetal hemoglobin, as well as GFP and hCD 122, asdescribed previously.

Example 18: Generation of Biologically Active TAT-Myc and TAT-Bcl-2Fusion Proteins

Fusion proteins having the HIV-1 TAT protein transduction domain (PTD)and either the ORF for human Myc, or a truncated form of human Bcl-2,that has been deleted for the unstructured loop domain (Anderson, M., etal. (1999). Prot Expr. Purif. 15, 162-70), were generated. Therecombinant proteins also encoded a V5 peptide tag and a 6-His tag, tofacilitate detection and purification (FIG. 18A). The amino acidsequence and nucleotide sequence of the TAT-MYC fusion protein aredepicted in FIG. 24. The amino acid sequence and nucleotide sequence ofthe TAT-Bcl-2Δ fusion protein are depicted in FIG. 25.

pTAT-Myc-V5-6×His (Amp^(R)) and pTAT-Bcl2 Δ-V5-6×His(Amp^(R)): plasmidwere generated by PCR amplification of a cDNA encoding human cMyc orhuman Bcl2 using a forward primer encoding an in frame TAT proteintransduction domain of HIV (RKKRRQRRR). The PCR products were clonedinto pET101/D-Topo (Invitrogen) vector. The unstructured loop (A.A.#27-80) was removed from the BCL-2 coding sequence using a Quick Changesite directed mutagenesis kit (Stratagene #200521-5).

The proteins were synthesized in E. coli and purified to homogeneity.SDS-PAGE electrophoresis and Coomassie Staining revealed the level ofpurity of the final product used for our studies (FIG. 18B).pTAT-Myc-V5-6×His was transformed into BL21-STAR(DE3) cells (Invitrogen)and protein was induced with 0.5 mM IPTG at 37° C. for 3 hrs. The cellswere lysed in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris pH to7.0, 10 mM imidazole, pH 7.2). The lysate was diluted to 6M urea andbrought to 450 mM NaCl, 50 mM NaH₂PO₄, 5 mM Tris pH 7.0. The lysate wastreated with Benzonase (500 units) at room temp for 1 hour, clarified bycentrifugation at 12,000 RPM for 60 min and filtered through a 0.22 μMfilter. Myc-V5-6×His was purified on a nickel affinity column (GE) usinga GE AKTA purifier 10 FPLC. Myc-V5-6×His was refolded by dialyzing intodialysis buffer (450 mM NaCl, 50 mM NaH₂PO₄, 5 mM Tris pH 7.0, 5%glycerol, 1 mM DTT). Endotoxin was reduced by passing the purifiedprotein over an Acticlean Etox column (Sterogen).

Bcl2Δ-V5-6×His protein was induced as described above. The cells werelysed in 50 mL of lysis buffer (200 mM NaCl, 200 mM KCL, 50 mM NaH₂PO₄,5 mM Tris pH 7.0, 5% glycerol, 1 mM DTT) supplemented with 500 unitsBenzonase, 1 mM PMSF, 2 ug/ml Leupeptin, 0.015 units/ml Aprotinin, 5 uMHen Egg Lysozyme (HEL) per 1 L of induced protein, and immediatelyplaced on ice for 1 hour. The cells were sonicated on ice (Dutycycle=50%, Output=5) for 2 sets of 2 minutes. The lysate was cleared bycentrifugation at 12,000 RPM for 60 min and was filtered through a 0.22μM filter. Bcl2Δ-V5-6×His was purified on a nickel affinity column (GE)and endotoxin was removed as described above.

Example 19: Confirmation of Appropriate Localization of TAT-FusionProteins

The fusion proteins localized to the appropriate intracellularcompartment (FIG. 18C). NIH 3T3 cells were seeded onto glass cover slipsin six-well plates and grown to 30 to 40% confluence. Each well wastransduced with 10 μg/ml of TAT-Myc or TAT-Bcl-2 or no treatment as anegative control. The cells were fixed in 4% paraformaldehyde-PBS for 10minutes at room temperature (RT) 2 hours following the proteintransduction. Cells were permeabilized in PBS supplemented with 1%bovine serum albumin (BSA) and 0.1% Triton X-100 at RT for 3 minutes.Cells were incubated for 45 minutes with V5 mouse monoclonal antiserum(Invitrogen) diluted in PBS-1% BSA (1:1,000). Cells were washed andincubated for 30 minutes with Goat anti-mouse Alexa 488 secondaryantibodies (Invitrogen A21121). Cover slips were mounted onto glassslides with a 10 μl drop of 50% glycerol with Hoechst at 1 μg/ml. Imageswere obtained on a Zeiss Imager Z1 Fluorescence microscope.

TAT-Myc rapidly localized to the nucleus in primary human HSCs (FIG.18D). TAT-fusion proteins are fully degraded after 72 hours in HSCs(FIG. 18E). Fetal cord blood cells were transduced with TAT-Myc andTAT-Bcl2Δ for 1 hour followed by 3 PBS washes. Two hourspost-transduction 5×10⁶ cells were harvested and the nuclear andcytoplasmic fractions were isolated. Cells (5×10⁶) were harvested every24 hours for the next 5 days. Nuclear and cytoplasmic proteins wereprepared by lysing cells in 10 mM HEPES (pH 7.6), 10 mM NaCl₂, 3 mMCaCl₂), and 0.5% NP40. Nuclei were pelleted, and thecytoplasmic-containing supernatant fraction was precipitated withtrichloroacetic acid (TCA). Following SDS-PAGE, Western blots wereprobed with anti-VS antibody (Invitrogen), anti-human β-actin (abcam),and goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (Santa CruzBiotechnology).

Example 20: Expansion of Human Cord Blood-Derived HSCs with TAT-Myc andTAT-Bcl-2

Fresh cord blood cells were obtained from samples that were discardedfrom a local cord blood bank. All human cells were de-identified andexempt from IRB oversight. Cord blood included O+, O−, A+, A−, B+, B−,and AB+ all of which showed approximately the same expansion profiles.

The total cord volume was split into 20 ml aliquots and diluted 1:1 inPBS. Diluted cord blood (20 mls) was gently overlaid on 20 mls ofFicoll-Paque Plus (Amersham Biosciences Cat #17-1440-03). The cells werespun at 900× gravity for 60 min. The huffy coat was removed with a glasspipette and was washed twice with PBS. The cells were resuspended in FCBmedia (Iscove's (Gibco) supplemented with 10% human plasma, 100 unitsper ml Penn/Strep, 30 ml of media containing SCF, IL3 and IL6 and 30 mlsof media containing TPO, FLT3-L, and GM-CSF described above. FCB mediawas further supplemented with 5 μg/ml recombinant TAT-Myc, and 10 μg/mlrecombinant TAT-Bcl-2 just prior to addition to the fetal cord blood(FCB) cells. The medium was replaced every 3 days over the course of theexpansion.

The cytokine cocktail contained IL3, IL6, TPO, Flt3-L, SCF, and GM-CSFwhich differs from previously reported media in the combination of thesesix cytokines (Suzuki, T., et al. (2006). Stem Cells 24, 2456-65.), aswell as by the addition of recombinant TAT-Myc and TAT-Bcl-2. Evaluationof the surface phenotype of the in vitro expanded human HSCs showed thatthe human HSCs retain their surface characteristics after extendedculture in the presence of TAT-Myc and TAT-Bcl-2 (FIG. 19A). This set ofconditions resulted in 86.4 fold increase in the number of CD34+ cellsin 14 days of culture, and 103.8 fold increase in the number of humanCD34+ cells derived from unfractionated cord blood in 21 days of culture(FIG. 19B).

Example 21: TAT-Myc and TAT-Bcl-2 Expanded Human CB HSCs areBiologically Active In Vitro and In Vivo

The in vitro expanded human HSCs were plated on MethoCult Optimum(StemCell Technologies), and were examined for their ability to giverise to specific colony types. The in vitro expanded human HSCs are ableto give rise to CFU-G, CFU-M, CFU-GM and BFU-E colonies (FIGS. 19C and19D). In addition, while the surface phenotype of the HSCs expanded inthe presence of TAT-Myc and TAT-Bcl-2 was preserved in culture, theircolony-forming unit content was significantly enriched under theseconditions (FIG. 19D). The CD34+ cells expanded in the presence ofTAT-Myc and TAT-Bcl-2 were also able to give rise to new BFU-E, CFU-M,CFU-G and CFU-GM colonies, whereas the CD34+ cells cultured in mediaalone did not generate new colonies (FIG. 19E).

NOD/SCID/gc−/− mice (NSG) mice were used as recipients for experimentsto test the ability of the human CD34+ cells expanded in vitro to giverise to mature human hematopoietic lineages in vivo. This is adocumented mouse model useful for this purpose (Tanaka, S., et al.(2012). Development of mature and functional human myeloid subsets inhematopoietic stem cell-engrafted NOD/SCID/IL2rgKO mice. J Immunol 188,6145-55.).

Fetal cord blood cells (FCBs) were injected into NOD/SCID/gc−/− mice(NSG) mice (Jackson Laboratory) that received 180 rads of radiation justprior to injection. Expanded FCBs were washed 3 times in PBS andinjected via the tail vein in 200μl PBS. Eight weeks post-transplant,the mice were bled via the tail vein to assess reconstitution by flowcytometry using the following antibodies: anti-human CD3 (hCD3)(Biolegend Cat #300312), anti-human CD19 (hCD19) (Biolegend Cat #302208)and anti-human CD45 (hCD45) (Biolegend Cat #304028).

Short term development of human CD45+ expressing T and B cells in NSGchimeric mice generated with 1×107 unfractionated cord blood cells wasobserved. However, the introduction of 1×106 protein-transducedlong-term (ptlt)-HSC generated in vitro by culture with TAT-Myc andTAT-Bcl-2 for 14 days resulted in a higher frequency of human CD45+cells in xenochimeric NSG mice. In addition, human CD45+ cells could beobserved in the peripheral blood of the mice for up to 20 weeks posttransplant (FIG. 20A). Human CD45+, CD34+CD3810 HSCs were found in thebone marrow (FIG. 20B), human CD45+/CD3+ and human CD45+/CD19+ lymphoidcells were found in the spleen, and human CD45+, CD3+ lymphoid cellswere found in the thymus of xenochimeric mice.

Human CD45+ CD19+ cells from the spleens of xenochimeric NSG mice werelabeled with CFSE, and were activated with monoclonal antibodies tohuman CD40 and IgM. The cells were analyzed at 72 hours by flowcytometry for dilution of CFSE. FIG. 20C shows the proliferation profileof the human B-cells that developed in vivo in xenochimeric NSG mice.

Human CD45+, CD34+CD3810 HSCs from the bone marrow of xenochimeric NSGmice were used to seed in MethoCult Optimum. These cells gave rise tocolonies in MethoCult plates (FIG. 20D), and some of the colonies couldstill be observed following serial replating (FIG. 20E). The number ofcolonies in both instances was significantly higher for NSG micereconstituted with human cord blood cells cultured for 14 days withTAT-Myc and TAT-Bcl-2 than for cells obtained from NSG micereconstituted with fresh, un-manipulated human cord blood cells.

In addition, a cohort of xenochimeric mice, engrafted with 106 cordblood cells previously expanded in vitro in a cocktail of cytokinessupplemented with TAT-Myc and TAT-Bcl-2 (black squares), were assessedfor myeloid and lymphoid cell differentiation. The CD45 positivepopulation of bone marrow cells (FIG. 20F) and spleen cells (FIG. 20G)were analyzed for CD11b, CD33, CD3, and CD19 expression. Both myeloidand lymphoid cell differentiation was observed in the bone marrow andspleen of these xenochimeric mice.

Example 22: Expansion of Human G-CSF Mobilized Peripheral Blood HSCswith TAT-Myc and TAT-Bcl-2

G-CSF mobilized cells were received in a 1 ml volume of elutriated bloodfrom 5 patients who underwent G-CSF mobilization for autologous HSCtransplantation. All G-CSF samples were de-identified and no furtheridentifying information was associated with the cells used for thesestudies. The cells were added drop wise to 10 ml of FCB media. The cellswere washed twice in FCB media and treated with 5 μg/ml recombinantTAT-Myc and 10 μg/ml recombinant TAT-Bcl-2 in a 10 ml volume. Cells(5×10⁶) were seeded in the G-Rex 100 cell expansion device (Wilson WolfManufacturing) according to the manufacturer's recommendation.

The cells were expanded in media supplemented with cytokines plusTAT-Myc and TAT-Bcl2 14 days. The FACS profile of the expanded HSCsshows a distinct population of hCD45+, CD34+, CD38hi, CD133+ cells (FIG.21A). The kinetics of cell expansion are illustrated in FIG. 21B.

The expanded adult GCS-F mobilized HSCs were then plated on MethoCultOptimum in order to characterize their differentiation potential invitro. The four colony types normally observed in the media thatsupports myeloerythroid differentiation were obtained (FIG. 21C), andsome of these colony types were also observed upon serial replating.

The expanded adult HSCs were able to reconstitute sublethally irradiatedNSG mice. FIG. 21D shows a FACS analysis of the CD45+ staining of bonemarrow from NSG mice transplanted 12 weeks earlier with either 106expanded G-CSF and TAT-Myc/TAT-Bcl-2 mobilized HSCs (first panel) or5×106 fresh un-manipulated cord blood cells (second panel).

The NSG xenochimeric mice generated with G-CSF mobilized cells culturedwith TAT-Myc and TAT-Bcl-2 were euthanized, and bone marrow, spleen andthymus were collected for further analysis. The analysis of lymphoidorgans from xenochimeric NSG mice reconstituted with expanded adult HSCsshowed that there were human CD45+, CD34+CD3810 cells in the bone marrow(FIG. 21E; first panel), human CD45+, CD3+ lymphoid cells in the spleen(FIG. 21E; second panel) and thymus (FIG. 21E; third panel) of thosemice. Together, these data demonstrate that one can successfully expandthe HSC population obtained from human G-CSF mobilized adult blood.

A cohort of xenochimeric mice engrafted with 10⁶ expanded G-CSFmobilized cells expanded in vitro in a cocktail of cytokinessupplemented with TAT-Myc and TAT-Bcl-2 (black squares) were assessedfor myeloid and lymphoid cell differentiation. The CD45 positivepopulation of bone marrow cells (FIG. 21F) and spleen cells (FIG. 21G)were analyzed for CD11b, CD33, CD3, and CD19 expression. Both myeloidand lymphoid cell differentiation was observed in the bone marrow andspleen of these xenochimeric mice.

This method is able to generate a sufficient number of HSCs needed fortransplantation of an average size adult according to current approaches(Sideri, A., et al. (2011). Hematologica 96, 1213-20.).

Example 23: Generation of Biologically Active Myc Fusion Proteins

Five Myc fusion proteins in addition to the TAT-Myc fusion proteindescribed in Example 18 were generated and purified using the sameapproach described there. The plasmids were made by PCR amplification ofthe coding region using a forward primer that contains an in frameN-terminal PTD-amino-acid sequence and a reverse primer that removed thestop codon. The PCR product was then cloned into pET101/D-Topo(Invitrogen) vector, which includes a C-terminal V5 epitope and6×-histidine purification tag. FIG. 22A shows a diagrammaticrepresentation of the Myc fusion proteins as compared with TAT-Myc fromExample 18. In each, a protein transduction (PTD) is fused in framebefore or after the Myc polypeptide.

Protein transduction domains included TAT, EPTD, and VPR. EPTD is anoptimized protein transduction domain (YARAAARQARA) taken from Ho, A. etal. (Synthetic protein transduction domains: enhanced transductionpotential in vitro and in vivo. Cancer Res. (2001) 61:474-477). VPRtransduction domain was as identified by Taguchi, T. et al. (Nucleartrafficking of macromolecules by an oligopeptide derived from VPR ofhuman immunodeficiency virus type-1. Biochem. Biophys. Res. Commun.(2004) 320(1):18-26).

Myc was either the ORF of the polypeptide as described in Example 1, orof the 3AMyc sequence previously described by Huang, Z. et al. (Negativecontrol of the Myc protein by the stress-responsive kinase Pak-2. MolCell Biol (2004) 24(4):1582-94). The recombinant proteins also encoded aV5 peptide tag and a 6-His tag, to facilitate detection andpurification. (FIG. 22A).

Example 24: Activated T Cell Survival Assays

The Myc fusion proteins described in Example 23 (TAT-Myc, TAT-3AMyc,EPTD-Myc, VPR-Myc, and Myc-VPR) were tested for Myc biological activityin an activated T cell viability assay (FIG. 22B). A spleen washarvested from a C57BL.6j (Jackson) mouse, and mechanically dissociatedthrough wire mess. The red blood cells were removed, and the T cellswere activated with 1 ug/ml anti-CD3 (2c11). The cells were plated intoa 24 well cluster dish at 3×10{circumflex over ( )}6 cells per well in 1ml of media. 48 hrs later, the live cells were captured on a Ficolcushion, washed, and plated in a 24 well cluster dishes at1-1.5×10{circumflex over ( )}6 cells per well. The PTD-Myc proteins weretitrated onto the T cells at 0.5, 1, 5, 10, 25, or 50 ug/ml. 48 hrsafter the PTD-Myc protein treatment, the cells were assessed forviability by flow cytometry (forward x side-scatter). In FIG. 22B, thedata presented are for the 25 ug/ml protein treatment.

As shown in FIG. 22B, all the constructs tested, except TAT-3AMyc,resulted in greater T cell viability after 48 hours than the untreatedcontrol. However, no construct resulted in greater T cell viability thanTAT-Myc described in Example 1.

In a similar experiment, the activity of TAT-Myc and TAT-Bcl-2 atvarious concentrations is shown in Table 5, below. T cells from spleensof C57BL.6j (Jackson) mice are activated with 1 ug/ml anti-CD3 (2c11).Following activation (48 hours later), the cells were washed, wereplated at about 1-1.5×10⁶ cells/well, and fusion proteins (TAT-Myc orTAT-Bcl-2) at various concentrations (0.5, 1, 5, 10, 25, or 50 ug/ml)were added. After 48 hours, the percent of live cells was determined byflow cytometry (forward x side scatter) as shown in Table 5, below.

TABLE 5 Concentration TAT-Bcl2 TAT-Myc [ug/ml] (% viable) (% viable) 08.5 3.1 0.5 9.5 5 1 11.4 7.68 5 21.1 14.3 10 22.4 24.4 25 31.9 25 5032.8 19.8

For both TAT-Myc and TAT-Bcl-2, and at all concentrations tested, cellviability and/or proliferation is increased as compared with cellsincubated in the absence of either fusion protein.

In a separate experiment using the same methods, FIG. 23 provides theFACS data for the live gate for activated T cells treated with 50 ug/mlof fusion proteins; TAT-Bcl-2 and TAT-Myc are compared with control(TAT-Cre or no treatment). As shown, both TAT-Myc and TAT-Bcl-2treatments result in significantly improved T cell survival and/orproliferation.

Example 25: Evaluation of Bcl-2

3T3 cells were transduced with TAT-Bcl2 for 1 hour followed by 3 PBSwashes. Two hours post-transduction, the cells were Trypsanized,counted, and 5×10⁶ were harvested. The nuclear and cytoplasmic fractionswere isolated. 5×10⁶ cells were harvested every 24 hours for the next 5days. Nuclear and cytoplasmic proteins were prepared by lysing cells in10 mM HEPES (pH 7.6), 10 mM NaCl₂, 3 mM CaCl₂), and 0.5% NP40. Nucleiwere pelleted, and the cytoplasmic-containing supernatant fraction wasprecipitated with trichloroacetic acid (TCA). Western blots were probedwith anti-VS antibody (Invitrogen), and goat anti-mouse IgG-HRP (SantaCruz Biotechnology).

TAT-Bcl2 was observed in the cytoplasmic fraction at 24 and 48 hours.The signal began to diminish by 72 hrs post transduction and was nolonger observed at the 96 hour time point.

Plasmids expressing TAT-Bcl2, TAT-Bcl2Δ, EPTD-Bcl2, VPR-Bcl2, VPR-Bcl2Δ,and VPR-BclXL were created. pPTD-Bcl2-V5-6×His(Amp^(R)): plasmids weregenerated by PCR amplification of a cDNA encoding human Bcl2 using aforward primer encoding an in frame PTD (TAT, EPTD or VPR) proteintransduction domain. The PCR products were cloned into pET101/D-Topo(Invitrogen) vector. To generate the Bcl2Δ the unstructured loop (A.A.#27-80) was removed from the BCL-2 coding sequence using a Quick Changesite directed mutagenesis kit (Stratagene #200521-5). VPR-BclXL was madein a similar fashion as the PTD-Bcl2 described above, but using the cDNAof human BclXL rather than Bcl2.

The amino acid sequence and nucleic acid sequence of TAT-Bcl-2Δ areshown in FIG. 25.

Example 26: Generation of Mature RBCs from HSCs

CD34+ cell were purified from mobilized peripheral blood using DynalCD34 positive selection beads according to the manufacturer's protocol.The CD34+ cells were treated for 1 hour at 37° C. with 5 ug/ml TAT-MYCand 5 ug/ml TAT-Bcl2 in iscoves media supplemented with 15% humanplasma, and 100 units/ml penn strep. Following the treatment withTAT-MYC and TAT-Bcl2, the cells are spun down at 1200 rpm for 5 minutesand the media is removed from the cell pellet. The treated primary CD34+cells are seeded in erythroid differentiation media (Iscoves mediasupplemented with 15% human plasma, 100 units/ml penn strep, 100units/ml EPO, and 3.2 ng/ml IL3) shifting their cellular programmingaway from being HSCs and towards erythroid cells because of the presenceof the 1L3 and EPO cytokines in the RBC media.

Treatin2 the purified CD34+ cells with this single bolus TAT-MYC andTAT-Bcl2 improves these differentiation cultures in 2 ways. First,primary CD34+ cell treated with TAT-fusion proteins show improvedviability during their differentiation resulting in the production of agreater number of mature red blood cells. Second, primary CD34+ celltreated with TAT-MYC and TAT-Bcl2 and seeded in RBC differentiationmedia show a better commitment to differentiating down the erythroidlineage rather then differentiating into other myeloid cells.

After the TAT-MYC and TAT-Bcl2 treatment, the CD34+ cells were seeded inwells of a 24 well cluster dish at 5×10{circumflex over ( )}4 cells perwell in RBC differentiation media described above. The cells wereallowed to differentiate for 11 days. On day 6 and 11, cells wereassessed for GPAxCD71 erythroid surface markers (FIG. 26A). The TAT-Mycand TAT-Bcl2 treated cells differentiate into erythroid cells asindicated by the 74% and 87.6% GPAxCD71 double positive cells (FIG.26.A; day 6 and 11 respectively). Additionally, these erythroid cellscontinue to express adult hemoglobin over fetal hemoglobin indicated bythe 78.8% of the cells that uniquely express adult hemoglobin comparedto the 19.8% of the cells that express fetal hemoglobin (FIG. 26A; day11 hBxfhB panel).

On day 10, a sample from the differentiation culture was cytospun on toa coverslip for H&E staining. Images are 10× and 20× magnification (FIG.26B). Hemoglobin expressing cells that have become anucleated wereobserved, as indicated by the red staining cells that lack a darkstaining nucleus in the center of the (FIG. 26B).

1.-65. (canceled)
 66. A method for treating a disease or disordercharacterized by a deficiency of anucleated red blood cells in a subjectin need thereof comprising: (a) culturing a population of hematopoieticstem cells in a differentiation media comprising erythropoietin (EPO)and a recombinant fusion protein comprising a MYC polypeptide fused to aprotein transduction domain, wherein culturing the hematopoietic stemcells induces differentiation of the hematopoietic stem cells to maturered blood cells, thereby producing a population of mature red bloodcells; and (b) administering to the subject the population of mature redblood cells.
 67. The method of claim 66, wherein the recombinant fusionprotein is TAT-MYC.
 68. The method of claim 66, wherein thedifferentiation media further comprises one or more recombinant proteinsthat inhibit apoptosis, wherein the one or more recombinant proteinsthat inhibit apoptosis comprises one or more Bcl2 homology domainsselected from the group consisting of BH1, BH2, BH3, and BH4.
 69. Themethod of claim 66, wherein the hematopoietic stem cells furthercomprise one or more recombinant proteins of interest.
 70. The method ofclaim 66, wherein the hematopoietic stem cells comprise one or moretransgenes that encode one or more recombinant proteins of interest. 71.The method of claim 70, wherein the expression or function of the one ormore recombinant proteins of interest is controllable or inducible. 72.The method of claim 66, wherein the disease or disorder characterized bya deficiency of anucleated red blood cells is selected from one or moreof anemia, Gaucher's disease, hemolysis, neutropenia, thrombocytopenia,granulocytopenia, hemophilia, Hodgkin's lymphoma, Non-Hodgkin'slymphoma, B cell chronic lymphoma, Burkitt's lymphoma, Follicular-likelymphoma, diffused large B-cell lymphoma, multiple myeloma, acutemyeloid leukemia, pre-B acute lymphocytic leukemia, pre-T acutelymphocytic leukemia, acute promyelocytic leukemia, or refractoryleukemia.
 73. The method of claim 72, wherein the disease or disordercharacterized by a deficiency of anucleated red blood cells is anemia.74. The method of claim 66, wherein the disease or disordercharacterized by a deficiency of anucleated red blood cells results fromone or more of chemotherapy, chemical exposure, radiation therapy,and/or radiation exposure.
 75. The method of claim 66, wherein thepopulation of mature red blood cells is co-administered with one or moreof chemotherapy, radiation therapy, or one or more proteins of interest.76. The method of claim 66, wherein the disease or disordercharacterized by a deficiency of anucleated red blood cells comprises aninfectious disease associated with hemorrhage.
 77. The method of claim76, wherein the infectious disease associated with hemorrhage comprisesa viral hemorrhagic fever.
 78. The method of claim 77, wherein the viralhemorrhagic fever is selected from Lassa fever, Ebola, Marburghemorrhagic fever, Rift Valley fever, dengue fever, or yellow fever. 79.The method of claim 66, wherein the population of mature red blood cellsexhibits one or more characteristics selected from the group consistingof: a population of mature red blood cells, wherein about 40% to about100% of the cells are anucleated; a population of mature red bloodcells, wherein about 40% to about 100% of the cells express glycophorinA (GPA); a population of mature red blood cells, wherein about 40% toabout 100% of the cells express adult hemoglobin; a population of maturered blood cells, wherein about 40% to about 100% of the cells exhibitdecreased levels of CD71 expression; and a population of mature redblood cells, wherein about 40% to about 100% of the cells exhibitdecreased levels of fetal hemoglobin expression.
 80. The method of claim66, wherein the population of hematopoietic stem cells is derived frombone marrow, peripheral blood, mobilized peripheral blood, umbilicalcord, placenta or fetal tissue, or is a hematopoietic cell line.
 81. Themethod of claim 66, wherein the subject is human.